Detection And Quantification Of Rare Variants With Low-depth Sequencing Via Selective Allele Enrichment Or Depletion

Abstract

This disclosure describes methods for enabling accurate detection and quantitation of rare alleles within a DNA sample using low-depth sequencing, through the use of allele-specific enrichment and/or depletion hybridization probes. For example, methods are provided for using competitive probes to apply allele-specific enrichment or depletion to amplicons from multiplex PCR on a biological DNA sample.

Description

REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. application Ser. No. 16/227,790, filed Dec. 20, 2018, which claims the priority benefit of U.S. Provisional Application No. 62/608,197, filed Dec. 20, 2017, the entire contents of each of which are incorporated herein by reference.

BACKGROUND

1. Field

[0003] The present disclosure relates generally to the field of molecular biology. More particularly, it concerns methods of enhancing detection of sequence variants by selective allele enrichment or depletion prior to sequencing by next-generation sequencing.

2. Description of Related Art

[0004] In biological samples, such as cell-free DNA from peripheral blood, rare DNA sequence variants, such as cancer driver mutations, are present at less than 1% allele frequency, but can nonetheless provide important therapy guidance or patient stratification information. Additionally, there is a need to simultaneously analyze many potential mutations to achieve high clinical sensitivity. Next-generation sequencing has been applied to detection and quantitation of rare DNA variants through deep sequencing with molecular barcodes. However, these methods are inherently inefficient and expensive due to the large number of NGS reads wasted on sequencing wildtype (i.e., healthy) DNA.

SUMMARY

[0005] The disclosure describes a class of methods to allow low-throughput detection and quantification of rare variants, such as somatic cancer mutations in peripheral blood plasma. The large number of reads needed for liquid biopsy applications prevents the detection and quantification of rare events by low-throughput NGS. However, allelic enrichment/depletion enables low-throughput NGS instruments, such as the Illumina MiSeq, the Qiagen GeneReader, and the Thermo Fisher Proton systems, to perform liquid biopsy detection of cancer mutations (see FIG. 6).

[0006] In one embodiment, provided herein are methods of detecting the presence of rare sequence variants within a DNA region of interest, the method comprising: (a) amplifying one or more region of interest using polymerase chain reaction (PCR) with primers, each primer comprising a 5' sequence-adaptor region and a 3' gene-specific region, thereby generating double-stranded amplicons; (b) denaturing the double-stranded amplicons, thereby generating single-stranded amplicons; (c) hybridizing the single-stranded amplicons to a mixture of negative-selection Sinks; (d) removing the single-stranded amplicons bound to Sinks; (e) amplifying the remaining single-stranded amplicons by PCR using primers comprising sequencing adaptor sequences; and (f) performing high-throughput DNA sequencing. In some aspects, the rare variant is of unknown sequence identity. In some aspects, the rare variant is of known sequence identity.

[0007] In some aspects, step (c) further comprises hybridizing the single-stranded amplicons to a mixture of positive-selection Probes. In certain aspects, the Probes comprise toehold probes, fine-tuned probes, or X-probes. In certain aspects, the Probes and Sinks are thermodynamically competitive. In some aspects, there is one Probe and one Sink for each rare sequence variant. In some aspects, there is one Probe for each rare sequence variant. In some aspects, two or more rare sequence variants may use the same Sink. In some aspects, the Probes comprise Probes having paired probe complement and probe protector oligonucleotides of Table 1. In some aspects, the Sink comprise Sinks having paired sink complement and sink protector oligonucleotides of Table 2. In certain aspects, step (d) further comprises collecting amplicons bound to Probes. In certain aspects, step (d) is performed via streptavidin-coated magnetic beads, collecting is performed using a magnet, and the Probes in step (c) are either directly functionalized with a biotin or hybridized to a universal oligonucleotide functionalized with a biotin. In certain aspects, step (d) is performed via streptavidin-coated agarose beads, collection is performed using centrifugal force, and the Probes in step (c) are either directly functionalized with a biotin or hybridized to a universal oligonucleotide functionalized with a biotin. In certain aspects, removing the single-stranded amplicons bound to Sinks occurs by way of collecting amplicons bound to Probes.

[0008] In some aspects, the hybridization in step (c) is performed at a temperature of between about 15.degree. C. and about 75.degree. C. In some aspects, the hybridization in step (c) is performed in a buffer with a monovalent cation concentration of between about 50 mM and about 5 M. In certain aspects, the monovalent cation is sodium. In some aspects, the hybridization in step (c) is performed in a buffer with a divalent cation concentration of between about 3 mM and about 30 mM. In certain aspects, the divalent cation is magnesium.

[0009] In some aspects, the PCR of step (a) is multiplex PCR when amplifying more than one region of interest. In some aspects, the PCR of step (a) is carried out for 4-20 cycles. In some aspects, the PCR of step (a) is carried out for no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cycles.

[0010] In some aspects, step (b) is performed via heat denaturation. In certain aspects, heat denaturation comprises heating the amplicon mixture to at least 80.degree. C. for at least 2 minutes. In some aspects, step (b) is performed via DNAse activity and wherein one of the primers in step (a) is modified with either a 5' phosphate functionalization to encourage degradation or a 5' functionalization to inhibit degradation. In certain aspects, the 5' primer functionalization comprises a phosphorothioate, a 2'-O-methyl group, or a non-natural nucleotide.

[0011] In some aspects, the Sinks in step (c) comprise toehold probes, fine-tuned probes, or X-probes. In some aspects, the removing in step (d) is performed via solid-phase separation. In some aspects, step (d) is performed via streptavidin-coated magnetic beads, removing is performed using a magnet, and the Sinks in step (c) are either directly functionalized with a biotin or hybridized to a universal oligonucleotide functionalized with a biotin. In some aspects, step (d) is performed via streptavidin-coated agarose beads, removing is performed using centrifugal force, and the Sinks in step (c) are either directly functionalized with a biotin or hybridized to a universal oligonucleotide functionalized with a biotin.

[0012] In some aspects, the primers in step (e) are universal primers. In some aspects, the primers in step (e) further comprise a sample barcode or index sequence. In some aspects, the sequencing in step (f) is sequencing-by-synthesis. In some aspects, the sequencing in step (f) is nanopore sequencing. In some aspects, the sequencing in step (f) is sequencing-by-hybridization (e.g., Nanostring).

[0013] In some aspects, the method further comprises (g) analyzing the DNA sequencing data to calculate the ratio of reads observed for variant sequences as compared to wild-type sequences. In some aspects, the sequencing in step (f) is paired-end sequencing. In some aspects, the analysis in step (g) does not consider any sequencing read in which the forward read and the reverse read do not perfectly agree on the sequence of the amplicon insert. In certain aspects, the analysis in step (g) does not consider any sequencing reading in which a read quality score is below 30. In certain aspects, the read quality score is a threshold FASTQ score.

[0014] In some aspects, the method is further defined as a method of quantifying the presence of rare sequence variants within a DNA region of interest.

[0015] As used herein, "essentially free," in terms of a specified component, means that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0016] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0017] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

[0018] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0019] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0022] FIG. 1: Example experimental workflow for enriching suspected genomic variants using positive-selection Probes and negative-selection Sinks. The concentration of gene-specific primers introduced in step 1 range between 5 and 80 nM. In step 2, the PCR protocol consists of an initial 98.degree. C. denaturation for 10 minutes, followed by 5 cycles of (98.degree. C. for 20 sec, 62.degree. C. for 4 min, and 72.degree. C. for 1 min) using the KAPA High Fidelity DNA polymerase. In step 4, the variant-specific Probes and wildtype-specific Sinks are introduced; the Probes are pre-hybridized to a universal biotinylated oligo whereas the Sinks are not. Both the Probes and the Sinks are double-stranded "toehold" or "fine-tuned" probes that each includes an auxiliary "Protector" oligonucleotide, which hybridizes to the Probe's complement strand. The stoichiometry of the Probe's protector varies between 1.5.times. and 9.times. relative to the Probe's complement strand. The stoichiometry of the Sink's protector varies between 2.times. and 11.times. relative to the Sink's complement strand. In step 7, the number of PCR cycles varies between 22 and 27 depending on sample input quantity and variant allele frequency, as well as prior expectations on the panel's fold enrichment. This PCR protocol consists of an initial 98.degree. C. denaturation for 10 minutes, followed by 5 cycles of (98.degree. C. for 20 sec, 62.degree. C. for 15 sec, and 72.degree. C. for 30 sec) using the KAPA High Fidelity DNA polymerase.

[0023] FIG. 2: Example of the computational workflow for analyzing sequencing results. This workflow is conservative in the sense that reads with any indication of error will be discarded, and only perfect reads will be used for assessing the allelic fraction of the Variant. Conservative analysis workflows tend to produce higher confidence on the allelic fraction at the cost of lower fraction of usable reads and sequencing depth.

[0024] FIGS. 3A-B: Experimental results for a 114-plex Variant enrichment panel using both positive and negative selection. The genomic DNA input sample consisted of 498.5 ng NA18537 cell line DNA and 1.5 ng NA18562 cell line DNA. The sample is thus 0.3% allele frequency in all single nucleotide polymorphisms (SNPs) in which both NA18537 and NA18562 are homozygous but differ from each other. The FIG. 3A graph shows that in a 2.2M read library, with roughly 10,000.times. depth per locus, there are roughly 30 variant reads per locus, as expected for the 0.3% allele frequency sample. For enrichment, Probes were designed to NA18562 SNP alleles and Sinks were designed to NA18537 alleles. The FIG. 3B graph shows that a 63 k read library produces similar reads for the variant for each locus, while the sequencing depth has been reduced 36-fold. Thus, sequencing cost can be reduced 36-fold while attaining similar information on rare mutations. See Tables 1-3 for sequences of the primers and probe oligonucleotides used to generate these data.

[0025] FIG. 4: Distribution of fold-enrichment per locus for the 114-plex panel summarized in FIG. 3. Median fold-enrichment observed was 52, and 90% of the Variants were enriched 8-fold or more. Fold-enrichment can be improved through empirical optimization of Probe or Sink sequence, or of Probe protector and Sink protector stoichiometry.

[0026] FIG. 5: Sequences for the Variant (SEQ ID NO: 685), Wild-type (SEQ ID NO: 686), Probe (SEQ ID NO: 6), Probe Protector (SEQ ID NO: 120), Universal Oligonucleotide Functionalized with Biotin (SEQ ID NO: 687), Sink (SEQ ID NO: 234), and Sink Protector (SEQ ID NO: 348) for one locus in the 114-plex panel. See Tables 1-3 for the full sequence list used for the 114-plex panel.

[0027] FIGS. 6A-B: Allele-selective enrichment sequencing (ASES). (FIG. 6A) Profiling rare mutations in cell-free DNA (cfDNA) requires extremely high sequencing depth as well as unique molecular identifier (UMI) barcodes. (FIG. 6B) ASES uses highly sequence-selective hybridization probes to enrich the variant allele fraction, allowing rare mutation profiling using low-depth sequencing.

[0028] FIGS. 7A-E: ASES sources of error and VAF limit of detection. (FIG. 7A) False positives arise from either PCR errors due to limited enzyme fidelity (e1) or NGS sequencing error (e0). (FIG. 7B) When using a pure human gDNA, all cancer mutations in the panel should have a VAF of 0%; non-zero VRF thus corresponds to the false positive error rate. (FIG. 7D) Overall distribution of ASES error rate in (FIG. 7C). (FIG. 7E) Per-locus error rate comparison between ASES and deep-sequencing. Error bar shows the standard deviation of the mean. Traces were ranked by mean per-locus error rate of ASES.

[0029] FIGS. 8A-D: Demonstration of ASES on an 118-plex non-pathogenic SNP panel. (FIG. 8A) Library preparation workflow. (FIG. 8B) NGS reads mapped to variant SNP alleles vs. wildtype alleles using standard deep sequencing. (FIG. 8C) NGS reads mapped to the variant SNP alleles vs. wildtype alleles using ASES. (FIG. 8D) Distribution of variant read fraction (VRF, variant reads divided by total reads mapped to each SNP locus) for standard deep sequencing vs. ASES.

[0030] FIGS. 9A-G: Estimation of sample VAF based on ASES VRF. (FIG. 9A) Consistent and predictable relationship between VRF, VAF, and fold-enrichment E. The dots represent experimental results from 7 different NGS libraries, where the input sample had known VAFs of 0.1%, 0.2%, 0.3%, 0.5%, 0.7%, 1%, 2%. The solid lines show theoretical curves for different values of E best-fitted to the three shown SNPs. The right panel plots VAF and VRF under non-linear transformations. (FIG. 9B) Distribution of r.sup.2 for the 118 SNP loci in the non-pathogenic ASES panel. One outlier SNP locus with r2<0 was omitted from the plot. (FIG. 9C) Distribution of best-t E. Error bars show the root mean square error (RMSE) of the linear t to the 7 data points for each SNP. Asterisk represents the one SNP in which the 0.1% VAF library did not have enough reads in the SNP locus to allow VRF quantitation; that SNP fitted E using the other 6 data points. (FIG. 9D) Distribution of fitted E for different base substitution types. One-way ANOVA indicates a p-value of 0.21, indicating that there is likely little to no sequence-based bias in E. (FIG. 9E) Accuracy of inferred VAF based on observed VRF and fitted E. (FIG. 9F) VAF quantitation based on VRF for standard deep sequencing. (FIG. 9G) NGS read uniformity across the 118 different amplicons, visualized by the cumulative distribution plots of NGS reads vs. number of loci (Lorenz curve).

[0031] FIGS. 10A-E: ASES actionable cancer mutation panel. (FIG. 10A) The distribution of mutations by corresponding cancer type, and the number of mutations profiled on each gene. (FIG. 10B) Distribution of potential cancer mutations on amplicons. (FIG. 10C) NGS reads mapped to cancer mutations vs. wildtype using standard deep sequencing (left) and ASES (right). (FIG. 10D) Distribution of observed fold-enrichment E, both in aggregate (left) and sorted by colocalization type (right). (FIG. 10E) Validation of cancer mutation panel on Horizon cfDNA reference samples (HD780 Multiplex I).

[0032] FIGS. 11A-B: Validation of the ASES cancer mutation panel on clinical cfDNA samples. (FIG. 11A) Summary of called mutations in 6 samples by deep sequencing, and in 64 samples by ASES. (FIG. 11B) Side-by-side comparison of ASES and deep sequencing on equal size aliquots of the same clinical cfDNA samples.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0033] The present disclosure describes methods for using toehold probes, fine-tune probes, or X-probes to apply allele-specific enrichment or depletion to amplicons from multiplex PCR on a biological DNA sample. Due to the high sequence specificity of the probes, a large majority of the wild-type sequences are removed, and the allele frequency of mutations are significantly increased. Consequently, low-depth sequencing becomes sufficient to detect and quantitate rare mutations. Thus, the industrial applicability of this disclosure is to significantly reduce sequencing costs for analyzing rare mutations.

[0034] Although these allele-specific enrichment and depletion probes have been previously demonstrated on DNA targets, integration with NGS is non-trivial. For example, direct application of toehold probes to biological DNA is undesirable because low probe capture yield and limited sample input quantity may result in false negatives, in which rare mutations are present in the original DNA sample but not captured by probes and consequently not represented in NGS data.

[0035] Furthermore, the current dominant method of NGS analysis of biological DNA is to end-repair fragmented genomic DNA and subsequently ligate to sequencing adaptors. However, end-repair and ligation are both low-yield enzymatic processes, and likewise can result in false negatives due to losing the few DNA molecules that bear a rare mutation.

[0036] Yet another possible but ultimately undesirable method is to perform many cycles of multiplexed amplification of gene regions of interest, and perform toehold probe enrichment or depletion on the final product. The drawback of this approach is that with high cycle multiplexed PCR, primer dimers become dominant, practically preventing this approach from scaling to more than 20 genetic loci.

[0037] The present disclosure thus describes the approach of performing low-cycle (e.g., 5) multiplex PCR to pre-amplify gene regions of interest by roughly 10- to 30-fold to counter probe binding loss, while simultaneously remaining scalable to high multiplexing due to the unlikelihood of accumulating high concentrations of primer dimers within only a few PCR cycles (see FIG. 1).

[0038] In some embodiments, both positive selection of the known rare variant is performed in combination with negative selection of the corresponding wild-type allele. In other embodiments, negative selection of wild-type alleles may be formed without concurrent positive selection, which allows for the detection of rare variants of unknown sequence.

[0039] ASES sources of error and VAF limit of detection. False positives arise from either PCR errors due to limited enzyme fidelity (e1) or NGS sequencing error (e0) (FIG. 7A). For example, when using a pure human gDNA, all cancer mutations in the panel should have a VAF of 0%; non-zero VRF thus corresponds to the false positive error rate (FIG. 7B). Median error rate of ASES was 0.610.quadrature.5, representing an almost 10-fold reduction in errors as compared to deep sequencing (FIGS. 7C-D). A per-locus error rate comparison between ASES and deep-sequencing is shown in FIG. 7E.

I. NUCLEIC ACID PROBES

[0040] In some embodiments, the present disclosure provides synthetic oligonucleotide probes for use in allele-specific enrichment and/or depletion. In particular embodiments, the oligonucleotide probes are toehold probes, X-probes, or fine-tune probes. The oligonucleotide probes can have a length of 30 to 200 nucleotides, particularly 50 to 100 nucleotides, such as between 60 and 70 nucleotides. Further, the oligonucleotide probes can comprise part or all of sequencing primer sequences or their binding sites, such as index sequencing primers for particular sequencing platforms (e.g., Illumina index primers).

[0041] The molecular specificity of the enrichment and depletion probes is beneficial to the accurate inference of genomic DNA variants. Nonspecific binding of variant enrichment probes to wild-type loci would defeat the purpose of enrichment. Likewise, nonspecific binding of wild-type depletion probes to variant loci would result in the desired target being lost from the sample. Toehold probes with protector oligonucleotides can be employed to enhance the molecular specificity of the Probes and Sinks. In some aspects, the toehold probes may be fine-tune probes as described in U.S. Pat. Publn. No. 2016/0340727, which is incorporated herein by reference in its entirety. In some aspects, the toehold probes may be X-probes as described in U.S. Pat. Publn. No. 2016/0326600, which is incorporated herein by reference in its entirety.

[0042] In some embodiments, a protector oligonucleotide comprising a region that is partially complementary to the target complementarity region is introduced. Importantly, at least five continuous nucleotides on the target complementarity region are not bound by the protector, i.e., form a toehold, in order to allow initiation of hybridization between the target and the Probe/Sink. This protector oligonucleotide can improve the specificity of hybridization reactions (see Zhang et al., 2012, Wang and Zhang, 2015, U.S. Pat. No. 9,284,602, and U.S. Pat. Publn. No. 2016/0340727, each of which is incorporated herein by reference in its entirety), and maintains high sequence selectivity across a large range of temperatures and buffer conditions. In some aspects, the protector oligonucleotide is present in molar excess.

[0043] In some embodiments, the nucleic acid probes are rationally designed so that the standard free energy for hybridization (e.g., theoretical standard free energy) between the specific target nucleic acid molecule and the target complementarity region is close to zero, while the standard free energy for hybridization between a spurious target (even one differing from the specific (actual) target by as little as a single nucleotide) and the probe is high enough to make their binding unfavorable by comparison.

[0044] The "toehold" region is present in the target complementarity region, is complementary to a target sequence and not complementary to the protector oligonucleotide. The sequences of the complementary regions are rationally designed to achieve this matching under desired conditions of temperature and probe concentration. As a result, the equilibrium for the actual target and Probe/Sink rapidly approaches 50% target:probe::protector:probe (or whatever ratio is desired), while equilibrium for the spurious target and primer greatly favors protector:probe.

[0045] Mechanistically, it is thought that hybridization to a target begins at the toehold and continues along the length of the target complementarity region until the probe is no longer "double-stranded." This assumes complementarity between the target and the target complementarity region. When nucleotide mismatches exist between a spurious target and the target complementarity region, displacement of the second strand (i.e., the protector oligonucleotide) is thermodynamically unfavorable and the association between the target complementarity region and the spurious target is reversed.

[0046] Because the standard free energy favors a complete match (fully complementary) between the target sequence of the nucleic acid and toehold regions of the probe rather than a mismatch (e.g., single nucleotide change), the target complementarity region of the probe will bind stably to a target in the absence of a mismatch but not in the presence of a mismatch. If a mismatch exists between the target complementarity region of the probe and the target, the probe duplex prefers to reform. In this way, the frequency of producing a ligation product when a target sequence is not present is reduced. This type of discrimination is typically not possible using the standard single-stranded probes because in those reactions there is no competing nucleic acid strand (such as the protector oligonucleotide) to which a mismatched probe strand would prefer to bind. In some aspects, the thermodynamics of the Probes and Sinks are designed to satisfy that of a Competitive Composition. See, U.S. Pat. Publn. No. US2017/0029875, which is incorporated herein by reference in its entirety.

[0047] In some aspects, the Sinks are functionalized with, for example, a biotin group to enable the removal of any target nucleic acids that are bound by the Sinks. In other aspects, the Probes are functionalized but the Sinks are not, thereby allowing any target nucleic acids bound by the Probes to be collected; in this aspect, the Sinks serve to compete with the Probes for binding to the non-desired targets to increase the specificity of the hybridization of the Probes.

[0048] In some embodiments, the sequence of the functionalized strand is decoupled from the sequence of the target-specific strand, such as, for example, in the case of X-probes. See, e.g., U.S. Pat. Publn. No. 2016/0326600, which is incorporated by reference herein in its entirety. In this embodiment, the probe system comprises a universal component and a target-specific component. The universal component comprises at least a first universal oligonucleotide/strand, which comprises at least one region. The sequence of the universal strand is not target specific and therefore can be used with any target-specific component. The target-specific component comprises a protector strand and a target-specific/complement strand. The target-specific strand (i.e. complement strand) comprises at least two regions. At least one of the regions of the target-specific strand is fully or partially complementary to the at least one region of the first universal strand, which gives rise to a first double-stranded region. In some instances, the protector strand has a region that is at least partially complementary (and in some instances fully complementary) to all or a portion of the target-specific strand, which gives rise to a second double-stranded region. The target-specific strand contains a toehold region that is not hybridized to any other strand of the probe, but is complementary to a portion of the target sequence. To be clear, the region of the target-specific strand that is complementary to a region of the protector strand is also complementary to the target sequence. In some embodiments, the first universal strand comprises a functionalization conjugated thereto.

[0049] Upon hybridization of the probe to the target nucleic acid, the protector strand and any universal strand hybridized thereto dissociates from the target-specific strand leaving the target-specific strand, along with any universal strand hybridized thereto, hybridized to the target nucleic acid. Thus, the probes of the present disclosure permit the use of functionalized universal components with a variety of target-specific components, thereby eliminating the expense of synthesizing a different functionalized probe for each desired target sequence.

[0050] In some aspects, the universal strands on the Sinks are functionalized with, for example, a biotin group to enable the removal of any target nucleic acids that are bound by the Sinks. In other aspects, the universal strands on the Probes are functionalized but the universal strands on the Sinks are not, thereby allowing any target nucleic acids bound by the Probes to be collected; in this aspect, the Sinks serve to compete with the Probes for binding to the non-desired targets to increase the specificity of the hybridization of the Probes.

II. FURTHER PROCESSING OF TARGET NUCLEIC ACIDS

[0051] A. Target Nucleic Acid Molecules

[0052] A nucleic acid molecule of interest can be a single nucleic acid molecule or a plurality of nucleic acid molecules. Also, a nucleic acid molecule of interest can be of biological or synthetic origin. Examples of nucleic acid molecules include genomic DNA, cDNA, RNA, amplified DNA, a pre-existing nucleic acid library, etc.

[0053] Nucleic acids in a nucleic acid sample being analyzed (or processed) in accordance with the present disclosure can be from any nucleic acid source. As such, nucleic acids in a nucleic acid sample can be from virtually any nucleic acid source, including but not limited to genomic DNA, complementary DNA (cDNA), RNA (e.g., messenger RNA, ribosomal RNA, short interfering RNA, microRNA, etc.), plasmid DNA, mitochondrial DNA, etc. Furthermore, as any organism can be used as a source of nucleic acids to be processed in accordance with the present disclosure, no limitation in that regard is intended. Exemplary organisms include, but are not limited to, plants, animals (e.g., reptiles, mammals, insects, worms, fish, etc.), bacteria, fungi (e.g., yeast), viruses, etc. In certain embodiments, the nucleic acids in the nucleic acid sample are derived from a mammal, where in certain embodiments the mammal is a human. A nucleic acid molecule of interest can be a single nucleic acid molecule or a plurality of nucleic acid molecules. Also, a nucleic acid molecule of interest can be of biological or synthetic origin. Examples of nucleic acid molecules include genomic DNA, cDNA, cell-free DNA (cfDNA), RNA, amplified DNA, a pre-existing nucleic acid library, etc. In some aspects, the target nucleic acid is a double-stranded DNA molecule, such as, for example, human genomic DNA.

[0054] A nucleic acid molecule of interest may be subjected to various treatments, such as repair treatments and fragmenting treatments. Fragmenting treatments include mechanical, sonic, chemical, enzymatic, degradation over time, etc. Repair treatments include nick repair via extension and/or ligation, polishing to create blunt ends, removal of damaged bases such as deaminated, derivatized, abasic, or crosslinked nucleotides, etc. A nucleic acid molecule of interest may also be subjected to chemical modification (e.g., bisulfite conversion, methylation/demethylation), extension, amplification (e.g., PCR, isothermal, etc.), etc.

[0055] An RNA molecule may be obtained from a sample, such as a sample comprising total cellular RNA, a transcriptome, or both; the sample may be obtained from one or more viruses; from one or more bacteria; or from a mixture of animal cells, bacteria, and/or viruses, for example. The sample may comprise mRNA, such as mRNA that is obtained by affinity capture. Obtaining nucleic acid molecules may comprise generation of the cDNA molecule by reverse transcribing the mRNA molecule with a reverse transcriptase, such as, for example Tth DNA polymerase, HIV Reverse Transcriptase, AMV Reverse Transcriptase, MMLV Reverse Transcriptase, or a mixture thereof.

[0056] B. Amplification of Nucleic Acids

[0057] A number of template-dependent processes are available to amplify the target nucleic acids present in a given sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR.TM.) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, each of which is incorporated herein by reference in its entirety. Briefly, two synthetic oligonucleotide primers, which are complementary to two regions of the template DNA (one for each strand) to be amplified, are added to the template DNA (that need not be pure), in the presence of excess deoxynucleotides (dNTP's) and a thermostable polymerase, such as, for example, Taq (Thermus aquaticus) DNA polymerase. In a series (typically 30-35) of temperature cycles, the target DNA is repeatedly denatured (around 90.degree. C.), annealed to the primers (typically at 50-60.degree. C.) and a daughter strand extended from the primers (72.degree. C.). As the daughter strands are created they act as templates in subsequent cycles. Thus, the template region between the two primers is amplified exponentially, rather than linearly.

[0058] A barcode, such as a sample barcode, may be added to the target nucleic acid molecules during amplification. One method involves annealing a primer to the target nucleic acid molecule, the primer including a first portion complementary to the target nucleic acid molecule and a second portion including a barcode; and extending the annealed primer to form a barcoded nucleic acid molecule. Thus, the primer may include a 3' portion and a 5' portion, where the 3' portion may anneal to a portion of the target nucleic acid molecule and the 5' portion comprises the barcode.

[0059] C. Sequencing of Nucleic Acids

[0060] Methods are also provided for the sequencing of the library of nucleic acid molecules. Any technique for sequencing nucleic acids known to those skilled in the art can be used in the methods of the present disclosure. DNA sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, sequencing-by-synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing-by-synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, and SOLiD sequencing.

[0061] The nucleic acid library may be generated with an approach compatible with Illumina sequencing such as a Nextera.TM. DNA sample prep kit, and additional approaches for generating Illumina next-generation sequencing library preparation are described, e.g., in Oyola et al. (2012). In other embodiments, a nucleic acid library is generated with a method compatible with a SOLiD.TM. or Ion Torrent sequencing method (e.g., a SOLiD.RTM. Fragment Library Construction Kit, a SOLiD.RTM. Mate-Paired Library Construction Kit, SOLiD.RTM. ChIP-Seq Kit, a SOLiD.RTM. Total RNA-Seq Kit, a SOLiD.RTM. SAGE.TM. Kit, a Ambion.RTM. RNA-Seq Library Construction Kit, etc.). Additional methods for next-generation sequencing methods, including various methods for library construction that may be used with embodiments of the present disclosure are described, e.g., in Pareek (2011) and Thudi (2012).

[0062] In particular aspects, the sequencing technologies used in the methods of the present disclosure include the HiSeq.TM. system (e.g., HiSeq.TM. 2000 and HiSeq.TM. 1000) and the MiSeq.TM. system from Illumina, Inc. The HiSeq.TM. system is based on massively parallel sequencing of millions of fragments using attachment of randomly fragmented genomic DNA to a planar, optically transparent surface and solid phase amplification to create a high density sequencing flow cell with millions of clusters, each containing about 1,000 copies of template per sq. cm. These templates are sequenced using four-color DNA sequencing-by-synthesis technology. The MiSeq.TM. system uses TruSeq.TM., Illumina's reversible terminator-based sequencing-by-synthesis.

[0063] Another example of a DNA sequencing platform is the QIAGEN GeneReader platform--a next generation sequencing (NGS) platform utilizing proprietary modified nucleotides whose 3' OH groups are reversely terminated by a small moiety to perform sequencing-by-synthesis (SBS) in a massively parallel manner. Briefly, the sequencing templates are first clonally amplified on a solid surface (such as beads) to generate hundreds of thousands of identical copies for each individual sequencing template, denaturized to generate single-stranded sequencing templates, hybridized with sequencing primer, and then immobilized on the flow cell. The immobilized sequencing templates are then subjected to a nucleotide incorporation reaction in a reaction mix that includes modified nucleotides with a cleavable 3' blocking group that enables the incorporation and detection of only one specific nucleotide onto each sequencing template in each cycle. See U.S. Pat. Nos. 6,664,079; 8,612,161; and 8,623,598, each of which is incorporated by reference herein.

[0064] Another example of a DNA sequencing platform is the Ion Torrent PGM.TM. sequencer (Thermo Fisher) and the Ion Torrent Proton.TM. Sequencer (Thermo Fisher), which are ion-based sequencing systems that sequence nucleic acid templates by detecting ions produced as a byproduct of nucleotide incorporation. Typically, hydrogen ions are released as byproducts of nucleotide incorporations occurring during template-dependent nucleic acid synthesis by a polymerase. The Ion Torrent PGM.TM. sequencer and Ion Proton.TM. Sequencer detect the nucleotide incorporations by detecting the hydrogen ion byproducts of the nucleotide incorporations. The Ion Torrent PGM.TM. sequencer and Ion Torrent Proton.TM. sequencer include a plurality of nucleic acid templates to be sequenced, each template disposed within a respective sequencing reaction well in an array. The wells of the array are each coupled to at least one ion sensor that can detect the release of H+ ions or changes in solution pH produced as a byproduct of nucleotide incorporation. The ion sensor comprises a field effect transistor (FET) coupled to an ion-sensitive detection layer that can sense the presence of H+ ions or changes in solution pH. The ion sensor provides output signals indicative of nucleotide incorporation, which can be represented as voltage changes whose magnitude correlates with the H+ ion concentration in a respective well or reaction chamber. Different nucleotide types are flowed serially into the reaction chamber, and are incorporated by the polymerase into an extending primer (or polymerization site) in an order determined by the sequence of the template. Each nucleotide incorporation is accompanied by the release of H+ ions in the reaction well, along with a concomitant change in the localized pH. The release of H+ ions is registered by the FET of the sensor, which produces signals indicating the occurrence of the nucleotide incorporation. Nucleotides that are not incorporated during a particular nucleotide flow will not produce signals. The amplitude of the signals from the FET may also be correlated with the number of nucleotides of a particular type incorporated into the extending nucleic acid molecule thereby permitting homopolymer regions to be resolved. Thus, during a run of the sequencer multiple nucleotide flows into the reaction chamber along with incorporation monitoring across a multiplicity of wells or reaction chambers permit the instrument to resolve the sequence of many nucleic acid templates simultaneously. Further details regarding the compositions, design and operation of the Ion Torrent PGM.TM. sequencer can be found, for example, in U.S. Pat. Publn. Nos. 2009/0026082; 2010/0137143; and 2010/0282617, all of which are incorporated by reference herein in their entireties.

[0065] Another example of a DNA sequencing technique that can be used in the methods of the present disclosure is 454 sequencing (Roche) (Margulies et al., 2005). 454 sequencing involves two steps. In the first step, DNA is sheared into fragments of approximately 300-800 base pairs, and the fragments are blunt ended. Oligonucleotide adaptors are then ligated to the ends of the fragments. The adaptors serve as primers for amplification and sequencing of the fragments. The fragments can be attached to DNA capture beads, e.g., streptavidin-coated beads using, e.g., Adaptor B, which contains 5'-biotin tag. The fragments attached to the beads are PCR amplified within droplets of an oil-water emulsion. The result is multiple copies of clonally amplified DNA fragments on each bead. In the second step, the beads are captured in wells (pico-liter sized). Pyrosequencing is performed on each DNA fragment in parallel. Addition of one or more nucleotides generates a light signal that is recorded by a CCD camera in a sequencing instrument. The signal strength is proportional to the number of nucleotides incorporated.

[0066] Another example of a DNA sequencing technique that can be used in the methods of the present disclosure is SOLiD technology (Life Technologies, Inc.). In SOLiD sequencing, genomic DNA is sheared into fragments, and adaptors are attached to the 5' and 3' ends of the fragments to generate a fragment library. Alternatively, internal adaptors can be introduced by ligating adaptors to the 5' and 3' ends of the fragments, circularizing the fragments, digesting the circularized fragment to generate an internal adaptor, and attaching adaptors to the 5' and 3' ends of the resulting fragments to generate a mate-paired library. Next, clonal bead populations are prepared in microreactors containing beads, primers, template, and PCR components. Following PCR, the templates are denatured and beads are enriched to separate the beads with extended templates. Templates on the selected beads are subjected to a 3' modification that permits bonding to a glass slide.

[0067] Another example of a DNA sequencing technique that can be used in the methods of the present disclosure is the IonTorrent system (Life Technologies, Inc.). Ion Torrent uses a high-density array of micro-machined wells to perform this biochemical process in a massively parallel way. Each well holds a different DNA template. Beneath the wells is an ion-sensitive layer and beneath that a proprietary Ion sensor. If a nucleotide, for example a C, is added to a DNA template and is then incorporated into a strand of DNA, a hydrogen ion will be released. The charge from that ion will change the pH of the solution, which can be detected by the proprietary ion sensor. The sequencer will call the base, going directly from chemical information to digital information. The Ion Personal Genome Machine (PGM.TM.) sequencer then sequentially floods the chip with one nucleotide after another. If the next nucleotide that floods the chip is not a match, no voltage change will be recorded and no base will be called. If there are two identical bases on the DNA strand, the voltage will be double, and the chip will record two identical bases called. Because this is direct detection--no scanning, no cameras, no light--each nucleotide incorporation is recorded in seconds.

[0068] Another example of a sequencing technology that can be used in the methods of the present disclosure includes the single molecule, real-time (SMRT.TM.) technology of Pacific Biosciences. In SMRT.TM., each of the four DNA bases is attached to one of four different fluorescent dyes. These dyes are phospholinked. A single DNA polymerase is immobilized with a single molecule of template single stranded DNA at the bottom of a zero-mode waveguide (ZMW). A ZMW is a confinement structure which enables observation of incorporation of a single nucleotide by DNA polymerase against the background of fluorescent nucleotides that rapidly diffuse in and out of the ZMW (in microseconds). It takes several milliseconds to incorporate a nucleotide into a growing strand. During this time, the fluorescent label is excited and produces a fluorescent signal, and the fluorescent tag is cleaved off. Detection of the corresponding fluorescence of the dye indicates which base was incorporated. The process is repeated.

[0069] A further sequencing platform includes the CGA Platform (Complete Genomics). The CGA technology is based on preparation of circular DNA libraries and rolling circle amplification (RCA) to generate DNA nanoballs that are arrayed on a solid support (Drmanac et al. 2010). Complete genomics' CGA Platform uses a novel strategy called combinatorial probe anchor ligation (cPAL) for sequencing. The process begins by hybridization between an anchor molecule and one of the unique adapters. Four degenerate 9-mer oligonucleotides are labeled with specific fluorophores that correspond to a specific nucleotide (A, C, G, or T) in the first position of the probe. Sequence determination occurs in a reaction where the correct matching probe is hybridized to a template and ligated to the anchor using T4 DNA ligase. After imaging of the ligated products, the ligated anchor-probe molecules are denatured. The process of hybridization, ligation, imaging, and denaturing is repeated five times using new sets of fluorescently labeled 9-mer probes that contain known bases at the n+1, n+2, n+3, and n+4 positions.

[0070] A further sequencing platform includes nanopore sequencing (Oxford Nanopore). Nanopore detection arrays are described in US2011/0177498; US2011/0229877; US2012/0133354; WO2012/042226; WO2012/107778, and have been used for nucleic acid sequencing as described in US2012/0058468; US2012/0064599; US2012/0322679 and WO2012/164270, all of which are hereby incorporated by reference. A single molecule of DNA can be sequenced directly using a nanopore, without the need for an intervening PCR amplification step or a chemical labelling step or the need for optical instrumentation to identify the chemical label. Commercially available nanopore nucleic acid sequencing units are developed by Oxford Nanopore (Oxford, United Kingdom). The GridION.TM. system and miniaturised MinION.TM. device are designed to provide novel qualities in molecular sensing such as real-time data streaming, improved simplicity, efficiency and scalability of workflows and direct analysis of the molecule of interest. Using the Oxford Nanopore nanopore sequencing platform, an ionic current is passed through the nanopore by setting a voltage across this membrane. If an analyte passes through the pore or near its aperture, this event creates a characteristic disruption in current. Measurement of that current makes it possible to identify the molecule in question. For example, this system can be used to distinguish between the four standard DNA bases G, A, T and C, and also modified bases. It can be used to identify target proteins, small molecules, or to gain rich molecular information, for example to distinguish between the enantiomers of ibuprofen or study molecular binding dynamics. These nanopore arrays are useful for scientific applications specific for each analyte type; for example when sequencing DNA, the technology may be used for resequencing, de novo sequencing, and epigenetics.

III. DEFINITIONS

[0071] "Amplification," as used herein, refers to any in vitro process for increasing the number of copies of a nucleotide sequence or sequences. Nucleic acid amplification results in the incorporation of nucleotides into DNA or RNA. As used herein, one amplification reaction may consist of many rounds of DNA replication. For example, one PCR reaction may consist of 30-100 "cycles" of denaturation and replication.

[0072] "Polymerase chain reaction," or "PCR," means a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors well-known to those of ordinary skill in the art, e.g., exemplified by the references: McPherson et al., editors, PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively).

[0073] "Primer" means an oligonucleotide, either natural or synthetic that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Usually primers are extended by a DNA polymerase. Primers are generally of a length compatible with its use in synthesis of primer extension products, and are usually are in the range of between 8 to 100 nucleotides in length, such as 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, 25 to 40, and so on, more typically in the range of between 18-40, 20-35, 21-30 nucleotides long, and any length between the stated ranges. Typical primers can be in the range of between 10-50 nucleotides long, such as 15-45, 18-40, 20-30, 21-25 and so on, and any length between the stated ranges. In some embodiments, the primers are usually not more than about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.

[0074] As used herein, a nucleic acid "region" or "domain" is a consecutive stretch of nucleotides of any length.

[0075] "Incorporating," as used herein, means becoming part of a nucleic acid polymer.

[0076] A "nucleoside" is a base-sugar combination, i.e., a nucleotide lacking a phosphate. It is recognized in the art that there is a certain inter-changeability in usage of the terms nucleoside and nucleotide. For example, the nucleotide deoxyuridine triphosphate, dUTP, is a deoxyribonucleoside triphosphate. After incorporation into DNA, it serves as a DNA monomer, formally being deoxyuridylate, i.e., dUMP or deoxyuridine monophosphate. One may say that one incorporates dUTP into DNA even though there is no dUTP moiety in the resultant DNA. Similarly, one may say that one incorporates deoxyuridine into DNA even though that is only a part of the substrate molecule.

[0077] "Nucleotide," as used herein, is a term of art that refers to a base-sugar-phosphate combination. Nucleotides are the monomeric units of nucleic acid polymers, i.e., of DNA and RNA. The term includes ribonucleotide triphosphates, such as rATP, rCTP, rGTP, or rUTP, and deoxyribonucleotide triphosphates, such as dATP, dCTP, dUTP, dGTP, or dTTP.

[0078] The term "nucleic acid" or "polynucleotide" will generally refer to at least one molecule or strand of DNA, RNA, DNA-RNA chimera or a derivative or analog thereof, comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine "A," guanine "G," thymine "T" and cytosine "C") or RNA (e.g. A, G, uracil "U" and C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide." "Oligonucleotide," as used herein, refers collectively and interchangeably to two terms of art, "oligonucleotide" and "polynucleotide." Note that although oligonucleotide and polynucleotide are distinct terms of art, there is no exact dividing line between them and they are used interchangeably herein. The term "adaptor" may also be used interchangeably with the terms "oligonucleotide" and "polynucleotide." In addition, the term "adaptor" can indicate a linear adaptor (either single stranded or double stranded) or a stem-loop adaptor. These definitions generally refer to at least one single-stranded molecule, but in specific embodiments will also encompass at least one additional strand that is partially, substantially, or fully complementary to at least one single-stranded molecule. Thus, a nucleic acid may encompass at least one double-stranded molecule or at least one triple-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a strand of the molecule. As used herein, a single stranded nucleic acid may be denoted by the prefix "ss," a double-stranded nucleic acid by the prefix "ds," and a triple stranded nucleic acid by the prefix "ts."

[0079] A "nucleic acid molecule" or "nucleic acid target molecule" refers to any single-stranded or double-stranded nucleic acid molecule including standard canonical bases, hypermodified bases, non-natural bases, or any combination of the bases thereof. For example and without limitation, the nucleic acid molecule contains the four canonical DNA bases--adenine, cytosine, guanine, and thymine, and/or the four canonical RNA bases--adenine, cytosine, guanine, and uracil. Uracil can be substituted for thymine when the nucleoside contains a 2'-deoxyribose group. The nucleic acid molecule can be transformed from RNA into DNA and from DNA into RNA. For example, and without limitation, mRNA can be created into complementary DNA (cDNA) using reverse transcriptase and DNA can be created into RNA using RNA polymerase. A nucleic acid molecule can be of biological or synthetic origin. Examples of nucleic acid molecules include genomic DNA, cDNA, RNA, a DNA/RNA hybrid, amplified DNA, a pre-existing nucleic acid library, etc. A nucleic acid may be obtained from a human sample, such as blood, serum, plasma, cerebrospinal fluid, cheek scrapings, biopsy, semen, urine, feces, saliva, sweat, etc. A nucleic acid molecule may be subjected to various treatments, such as repair treatments and fragmenting treatments. Fragmenting treatments include mechanical, sonic, and hydrodynamic shearing. Repair treatments include nick repair via extension and/or ligation, polishing to create blunt ends, removal of damaged bases, such as deaminated, derivatized, abasic, or crosslinked nucleotides, etc. A nucleic acid molecule of interest may also be subjected to chemical modification (e.g., bisulfite conversion, methylation/demethylation), extension, amplification (e.g., PCR, isothermal, etc.), etc.

[0080] Nucleic acid(s) that are "complementary" or "complement(s)" are those that are capable of base-pairing according to the standard Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules. As used herein, the term "complementary" or "complement(s)" may refer to nucleic acid(s) that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above. The term "substantially complementary" may refer to a nucleic acid comprising at least one sequence of consecutive nucleobases, or semiconsecutive nucleobases if one or more nucleobase moieties are not present in the molecule, are capable of hybridizing to at least one nucleic acid strand or duplex even if less than all nucleobases do not base pair with a counterpart nucleobase. In certain embodiments, a "substantially complementary" nucleic acid contains at least one sequence in which about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, to about 100%, and any range therein, of the nucleobase sequence is capable of base-pairing with at least one single or double-stranded nucleic acid molecule during hybridization. In certain embodiments, the term "substantially complementary" refers to at least one nucleic acid that may hybridize to at least one nucleic acid strand or duplex in stringent conditions. In certain embodiments, a "partially complementary" nucleic acid comprises at least one sequence that may hybridize in low stringency conditions to at least one single or double-stranded nucleic acid, or contains at least one sequence in which less than about 70% of the nucleobase sequence is capable of base-pairing with at least one single or double-stranded nucleic acid molecule during hybridization.

[0081] The term "non-complementary" refers to nucleic acid sequence that lacks the ability to form at least one Watson-Crick base pair through specific hydrogen bonds.

[0082] The term "ligase" as used herein refers to an enzyme that is capable of joining the 3' hydroxyl terminus of one nucleic acid molecule to a 5' phosphate terminus of a second nucleic acid molecule to form a single molecule. The ligase may be a DNA ligase or RNA ligase. Examples of DNA ligases include E. coli DNA ligase, T4 DNA ligase, and mammalian DNA ligases.

[0083] "Sample" means a material obtained or isolated from a fresh or preserved biological sample or synthetically-created source that contains nucleic acids of interest. In certain embodiments, a sample is the biological material that contains the variable immune region(s) for which data or information are sought. Samples can include at least one cell, fetal cell, cell culture, tissue specimen, blood, serum, plasma, saliva, urine, tear, vaginal secretion, sweat, lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascites fluid, fecal matter, body exudates, umbilical cord blood, chorionic villi, amniotic fluid, embryonic tissue, multicellular embryo, lysate, extract, solution, or reaction mixture suspected of containing immune nucleic acids of interest. Samples can also include non-human sources, such as non-human primates, rodents and other mammals, other animals, plants, fungi, bacteria, and viruses.

[0084] As used herein in relation to a nucleotide sequence, "substantially known" refers to having sufficient sequence information in order to permit preparation of a nucleic acid molecule, including its amplification. This will typically be about 100%, although in some embodiments some portion of an adaptor sequence is random or degenerate. Thus, in specific embodiments, substantially known refers to about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

IV. KITS

[0085] The technology herein includes kits for creating libraries of target nucleic acids in a sample. A "kit" refers to a combination of physical elements. For example, a kit may include, for example, one or more components, such as Sinks and Probes, either with or without protector oligonucleotides, as well as specific primers, enzymes, reaction buffers, an instruction sheet, and other elements useful to practice the technology described herein. These physical elements can be arranged in any way suitable for carrying out the disclosure.

[0086] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted (e.g., aliquoted into the wells of a microtiter plate). Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a single vial. The kits of the present disclosure also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0087] A kit will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented. It is contemplated that such reagents are embodiments of kits of the disclosure. Such kits, however, are not limited to the particular items identified above.

V. EXAMPLES

[0088] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

Example 1--Allele Selective Enrichment Sequencing with 114-plex Non-pathogenic Panel

[0089] A 114-plex non-pathogenic panel has been completed using both positive and negative selection. See Tables 1-3 for the full sequence list used for the 114-plex panel. See FIG. 5 for an illustration of the sequences for the Variant, Wild-type, Probe, and Sink for one locus in the panel.

[0090] The genomic DNA input sample consisted of 498.5 ng NA18537 cell line DNA and 1.5 ng NA18562 cell line DNA. The sample had a 0.3% allele frequency in all single nucleotide polymorphisms (SNPs) in which both NA18537 and NA18562 are homozygous but differ from each other. In a 2.2M read library, with roughly 10,000.times. depth per locus, there are roughly 30 variant reads per locus, as expected for the 0.3% allele frequency sample (FIG. 3A). For enrichment, Probes were designed to NA18562 SNP alleles and Sinks were designed to NA18537 alleles. A 63 k read library produces similar reads for the variant for each locus, while the sequencing depth has been reduced 36-fold (FIG. 3B). Thus, sequencing cost can be reduced 36-fold while attaining similar information on rare mutations.

[0091] The distribution of fold-enrichment per locus for the 114-plex panel was analyzed. Median fold-enrichment observed was 52, and 90% of the Variants were enriched 8-fold or more (FIG. 4). Fold-enrichment can be improved through empirical optimization of Probe or Sink sequence, or of Probe protector and Sink protector stoichiometry.

[0092] In addition, panels using only negative selection, in which only wild-type alleles are depleted and thus the sequence of the variant need not be known, is also contemplated.

Example 2--Allele Selective Enrichment Sequencing with 118-plex Non-pathogenic Panel

[0093] Amplicons were hybridized to variant-specific probes and wildtype-specific sinks to enrich variants of interest (FIG. 8A). Probes and sinks were implemented as partially double-stranded toehold probes, following thermodynamics and kinetics principles described in (Zhang, 2012). Probes were hybridized to universal biotinylated oligos to allow magnetic bead-based separation of amplicons bound to the probes. NGS reads were mapped to variant SNP alleles vs. wildtype alleles using standard deep sequencing. The input was 30 ng of a 0.2%:99.8% mixture of two human genomic DNA (gDNA) samples, NA18562 and NA18537. For each of the 118 SNPs in the panel, NA18562 and NA18537 are homozygous for different alleles; here the NA18537 alleles were considered the wildtype alleles, so the sample was 0.2% variant allele frequency (VAF) for all 118 SNPs. NGS results were consistent with expectations (FIG. 8B). NGS reads were mapped to the variant SNP alleles vs. wildtype alleles using ASES. The median ratio of variant reads to wildtype reads was 22.4%, indicating minor allele enrichment by a factor of roughly 100 over standard deep sequencing shown in FIG. 8B (FIG. 8C). FIG. 8D provides the distribution of variant read fraction (VRF, variant reads divided by total reads mapped to each SNP locus) for standard deep sequencing vs. ASES.

[0094] Estimation of sample VAF based on ASES VRF. FIG. 9A shows the consistent and predictable relationship between VRF, VAF, and fold-enrichment E. The dots represent experimental results from 7 different NGS libraries, where the input sample had known VAFs of 0.1%, 0.2%, 0.3%, 0.5%, 0.7%, 1%, 2%. The solid lines show theoretical curves for different values of E best-fitted to the three shown SNPs. The right panel plots VAF and VRF under non-linear transformations. The linear correlation constant r.sup.2 of log.sub.10(f(VRF)) vs. log.sub.10(f(VAF)) indicates both confidence in the fitted parameter E and accuracy in inferring VAF from VRF. FIG. 9B shows the distribution of r.sup.2 for the 118 SNP loci in the non-pathogenic ASES panel. One outlier SNP locus with r.sup.2<0 was omitted from the plot. FIG. 9C shows the distribution of best-t E. Error bars show the root mean square error (RMSE) of the linear t to the 7 data points for each SNP. Asterisk represents the one SNP in which the 0.1% VAF library did not have enough reads in the SNP locus to allow VRF quantitation; that SNP fitted E using the other 6 data points. FIG. 9D shows the distribution of fitted E for different base substitution types. One-way ANOVA indicates a p-value of 0.21, indicating that there is likely little to no sequence-based bias in E. FIG. 9E looks at the accuracy of inferred VAF based on observed VRF and fitted E. Here, leave-one-out was performed: E was tted to 6 out of 7 data points each SNP, and used to calculate the VAF of the last data point based on the VRF. VAF quantitation based on VRF for standard deep sequencing is shown in FIG. 9F. VAF quantitation accuracy was similar for deep sequencing and ASES. FIG. 9G shows the NGS read uniformity across the 118 different amplicons, visualized by the cumulative distribution plots of NGS reads vs. number of loci (Lorenz curve). The Gini index G denotes double the area between the cumulative distribution plot and the perfect equality dotted line (G=0 indicates perfect equality and G=1 indicates perfect inequality where all reads are in a single locus). The value of G for deep sequencing and ASES were observed to be similar.

Example 3--Allele Selective Enrichment Sequencing with 112-plex Cancer Mutation Panel

[0095] A cancer mutation panel, based on the National Comprehensive Cancer Network (NCCN) guidelines of actionable mutations, was generated. The panel covers 112 actionable mutations distributed across 42 amplicons (FIG. 10A). Shown are the distribution of mutations by corresponding cancer type, and the number of mutations profiled on each gene. FIG. 10B shows the distribution of potential cancer mutations on amplicons. Type 1, isolated mutations, are similar to the SNPs profiled in the NGS panel shown in FIGS. 9A-G. Type 2, sparse mutations, have multiple mutations colocalized on the same amplicon, but each mutation is sufficiently far from other mutations that we do not expect any interference between probes and sinks to each mutation. Type 3, clustered mutations, have multiple mutations all colocalized within a small window, so that the same wildtype sink can be applied to all of the mutations in the cluster. Type 4, complex groups, have clusters of mutations that are not sufficiently close to each other to be covered by the same wildtype sink, and require more complex and suboptimal design of probes and sinks. FIG. 10C shows NGS reads mapped to cancer mutations vs. wildtype using standard deep sequencing (left) and ASES (right). Here, the input was 30 ng of human genomic DNA (NA18537), spiked in with synthetic DNA molecules bearing each of the profiled mutations at 0.1% VAF. Note that because multiple hotspot mutations share the same wildtype, the same wildtype reads are repeatedly plotted at multiple different mutation loci; these wildtype reads are counted only once when considering total reads. FIG. 10D shows the distribution of observed fold-enrichment E, both in aggregate (left) and sorted by colocalization type (right). The mutation colocalization type appears to significantly affect E, with type 1 being the best performers and type 4 being the worst performers. FIG. 10E shows the validation of the cancer mutation panel on Horizon cfDNA reference samples (HD780 Multiplex I). Inferred VAF from ASES generally agreed with nominal VAFs provided by Horizon product insert.

[0096] Validation of the ASES cancer mutation panel on clinical cfDNA samples is shown in FIGS. 11A-B. FIG. 11A provides a summary of called mutations in 6 samples by deep sequencing, and in 64 samples by ASES. Due to the reduced NGS reads required, all 64 samples for ASES were profiled using a single MiSeq run, while the 6 samples for deep sequencing required 2 separate MiSeq chips. Mutations were called for the ASES panel if the inferred VAF was more than 3 standard deviations greater than the median error rate, on a per-locus basis. Mutations were called for the deep sequencing panel if the inferred VAF was more than double the error rate, on a per-locus basis. A significant number of cancer mutations were found to be present even in cfDNA from healthy donors at between 0.01% and 1% inferred VAF. FIG. 11B provides a side-by-side comparison of ASES and deep sequencing on equal size aliquots of the same clinical cfDNA samples.

TABLE-US-00001 TABLE 1 Exemplary Probes SNP Probe Complement Probe Protector rs2246745 AAATAATCAGGAGAAGGAGATGGCATGTTTGTTGG TGACTGAGAGAGCTCCTTGGAATCACCAACAAACA TGATTCCAAGGAGCTCTCTCAGTCATGATcctgtacatttg TGCCATCTCCT (SEQ ID NO: 115) ctctgcctt (SEQ ID NO: 1) rs1805105 GGAGCAGCGTCTCTGCCATCGTCCTCGTCCATGTCC AAGTAGTTAATCTGGAAGTGTGACCAGGACATGGA TGGTCACACTTCCAGATTAACTACTTCGAAcctgtacattt CGAGGACGATGGCAGAGA (SEQ ID NO: 116) gctctgcctt (SEQ ID NO: 2) rs3789806 AGGTAAATATTTACCACCTCTTGGTGTTTATTTTAC cGGTGCTCTTGTATATAGACGGTAAAATAAACACCA CGTCTATATACAAGAGCACCGCAAcctgtacatttgctctgcct AGAGGT (SEQ ID NO: 117) t (SEQ ID NO: 3) rs9648696 CTTTCAGTCAGATGTATATGCATTTGGGATTGTTCT GCACATACTCATCAATTCATACAGAACAATCCCAA GTATGAATTGATGAGTATGTGCGGTTcctgtacatttgctctg ATGCATATACA (SEQ ID NO: 118) cctt (SEQ ID NO: 4) rs116952709 TATCTGCTAAGAAACAGACATCCATATACAGAGAT TGATGTATCAAAAATCATCATTTTCATCTCTGTATA GAAAATGATGATTTTTGATACATCAACTGcctgtacatttg TGGATGTCTGT (SEQ ID NO: 119) ctctgcctt (SEQ ID NO: 5) rs2511854 ATCTTGCCTTGCCTTCCACCCTAATACCAGCATAAT TCAGTTGTAGAGAAGCTTTAGTAGATTATGCTGGTA CTACTAAAGCTTCTCTACAACTGATACGcctgtacatttgct TTAGGGTGGAAGG (SEQ ID NO: 120) ctgcctt (SEQ ID NO: 6) rs2510152 TGTCCAGTGATATGGTTATAATGTGAAACAAAACT TCTGCAGTAAAAGTGACCCAGGTGAGTTTTGTTTCA CACCTGGGTCACTTTTACTGCAGACCTTcctgtacatttgct CATTATAACCA (SEQ ID NO: 121) ctgcctt (SEQ ID NO: 7) rs2066827 ATTAAAGGCGCCGCCGGGCGGCTCCCGCTGCCATC TCTCAGTGCGCAGGAGAGCCAGGATGGCAGCGGGA CTGGCTCTCCTGCGCACTGAGAGATAAcctgtacatttgctc GCCGCCCGGCG (SEQ ID NO: 122) tgcctt (SEQ ID NO: 8) rs129974 GATTTGCGTTCTGCACTATGACATAATTTGGCCTTC ATATATATCGGAAAACAACTCACCCTGAAGGCCAA AGGGTGAGTTGTTTTCCGATATATATGTCTcctgtacattt ATTATGTCATAGTGCA (SEQ ID NO: 123) gctctgcctt (SEQ ID NO: 9) rs2228422 AGACTTCACCTTGTGATCTGCAGGGACTGACCTTAG AGACTATTGCCAACACAACAACACTAAGGTCAGTC TGTTGTTGTGTTGGCAATAGTCTTGGAcctgtacatttgctct CCTGCAGATCACA (SEQ ID NO: 124) gcctt (SEQ ID NO: 10) rs3738807 AGAGATCTCCAAAGACACTCCACGGAATGAGGGCT TGATATTAACATAAAGACAAGGGCAACAAGCCCTC TGTTGCCCTTGTCTTTATGTTAATATCAACAGcctgtac ATTCCGTGGAGTGTCT (SEQ ID NO: 125) atttgctctgcctt (SEQ ID NO: 11) rs2294976 CCTATACAATTGAGATGTTGGGGGAACCACAACAT GCTCGTCACAGTCTGAATGAGTTATGTTGTGGTTCC AACTCATTCAGACTGTGACGAGCTTGAcctgtacatttgctc CCCAACAT (SEQ ID NO: 126) tgcctt (SEQ ID NO: 12) rs2305351 TTTTTATTGTATATGCATGCACATCCCAAGGACCAA CACATGCGGTGTAGCTGGTCTCTTGGTCCTTGGGAT GAGACCAGCTACACCGCATGTGACGAcctgtacatttgctct GTGCATG (SEQ ID NO: 127) gcctt (SEQ ID NO: 13) rs1630312 GCAGGCATCAAAGTGCAGGACGTCCGGCTGAATGG TGGCAGCTTTGGCCAGCTGCGGAGCCATTCAGCCG CTCCGCAGCTGGCCAAAGCTGCCATTCactgtacatttgct GACGTCCTGCACTT (SEQ ID NO: 128) ctgcctt (SEQ ID NO: 14) rs10873531 TCTGCATTCCCTGTCACTGCGTCACTGGCCTTCAGA TAGCAATCAAGCACCTTGGCTCTGTCTGAAGGCCA CAGAGCCAAGGTGCTTGATTGCTACAATcctgtacatttgc GTGACGCAGTGACAG (SEQ ID NO: 129) tctgcctt (SEQ ID NO: 15) rs8005905 GCCCAAGTGTTTCTCTGGCATCTGTTGGTGTCTGGA GAAGCACCAGTAGAGTGGTGGATCCAGACACCAAC TCCACCACTCTACTGGTGCTTCGTCAcctgtacatttgctctgc AGATGCCAGAGAA (SEQ ID NO: 130) ctt (SEQ ID NO: 16) rs117396186 GTTCAGATTTCACTGCCTCATGTTGATATTTCTTTCC GTGAGAGATTAAACAGTCGTCATCTGGAAAGAAAT AGATGACGACTGTTTAATCTCTCACACCTcctgtacatttg ATCAACATGAGGCA (SEQ ID NO: 131) ctctgcctt (SEQ ID NO: 17) rs34937835 TAGACACATTGTCATCATGGACAGGCGGTGGATAC ATAGTTAGGTATCAGCTAGACGGCACGTATCCACC GTGCCGTCTAGCTGATACCTAACTATAGAAcctgtacatt GCCTGTCCATGATG (SEQ ID NO: 132) tgctctgcctt (SEQ ID NO: 18) rs17224367 GCTTGTCTTTGAAACTTCTTGGCAAATCGGTTAAGA AGGTATGAACCGTCGATTCCCAGATCTTAACCGATT TCTGGGAATCGACGGTTCATACCTCAATcctgtacatttgct TGCCAAGAAGTTT (SEQ ID NO: 133) ctgcctt (SEQ ID NO: 19) rs2303428 TACCTCCCATATTGGGGCCTACAGAACAAATTATAT CGGTATCAATCTTGCTTTCTGATATAATTTGTTCTGT CAGAAAGCAAGATTGATACCGTTGAcctgtacatttgctctgc AGGCCCCA (SEQ ID NO: 134) ctt (SEQ ID NO: 20) rs2229910 TGCCAATGACCACAGTGTCGGGCCCCGCATCCAGT GTTCTCCCACCACGCCCTCGTCACTGGATGCGGGGC GACGAGGGCGTGGTGGGAGAACCAGAcctgtacatttgctct CCGACACTGTG (SEQ ID NO: 135) gcctt (SEQ ID NO: 21) rs200267496 ATCCCCCTTAAAATCACGCTCACTTGCCGCGCATAG tTCGTCGTCAACATCGAGATGGCCTATGCGCGGCAA GCCATCTCGATGTTGACGACGAACAGcctgtacatttgctctg GTGAGCGTGAT (SEQ ID NO: 136) cctt (SEQ ID NO: 22) rs17334387 GGGGAGGTGAAGCTGTCTATCTCCTACAAAAACAA TTGACGTTAATATGATGAAGAGTTTATTGTTTTTGT TAAACTCTTCATCATATTAACGTCAATAAAcctgtacattt AGGAGATAGACAGCTT (SEQ ID NO: 137) gctctgcctt (SEQ ID NO: 23) rs706713 CGAGATTTTTTTCCTTCCAATATATTCTACATAAGT TAGATCCATTCTGGGACTTTCCGGGAACTTATGTAG TCCCGGAAAGTCCCAGAATGGATCTAAAAGcctgtacat AATATATTGGAAG (SEQ ID NO: 138) ttgctctgcctt (SEQ ID NO: 24) rs706714 CTTTTCCTTAAAAAGAAAAAGAAAGGGAGTCATTA ATCGGTGATACGTGGTTGCTTAATGACTCCCTTTCT AGCAACCACGTATCACCGATCATTGTcctgtacatttgctctg TTTTC (SEQ ID NO: 139) cctt (SEQ ID NO: 25) rs290223 TGTGTGTTATGATTTCTGTTGCAGAGTTGTGAAAAC ACCTCTGGTGTTTTCACAGCCACGGTTTTCACAACT CGTGGCTGTGAAAACACCAGAGGTCTTCcctgtacatttgc CTGCAACAGAA (SEQ ID NO: 140) tctgcctt (SEQ ID NO: 26) rs1230345 GGCCCTGCAAATGCCCTCATCAGAAGCCCCGTTGC TCTTCGAGTGCTCACTCCAGGAGGGCAACGGGGCT CCTCCTGGAGTGAGCACTCGAAGATCGAcctgtacatttgc TCTGATGAGGGCATTT (SEQ ID NO: 141) tctgcctt (SEQ ID NO: 27) rs16754 GCTACTCCAGGCACACGCCGCACATCCTGCAGGCA CTGTATCGACTTCCTCTTACTCTCTGCCTGCAGGAT GAGAGTAAGAGGAAGTCGATACAGCCTAcctgtacatttg GTGCGGCGTGTGC (SEQ ID NO: 142) ctctgcctt (SEQ ID NO: 28) rs6667687 GATCTCTCCTGAGTCCTCACTAACAACAGGGGGTA ACTTGGCTTGAAAACAATAAATCTACCCCCTGTTGT GATTTATTGTTTTCAAGCCAAGTATCAcctgtacatttgctct TAGTGAGGAC (SEQ ID NO: 143) gcctt (SEQ ID NO: 29) rs3737639 CCACTAGCCCTGGTTCAGGTCAGGGATGCCATGTC AAGATTTGCCTGGGCCCCGACGACATGGCATCCCT GTCGGGGCCCAGGCAAATCTTTTCGcctgtacatttgctctgc GACCTGAACCA (SEQ ID NO: 144) ctt (SEQ ID NO: 30) rs880724 TGGCAGCCTCACTGTGCGGAGCATGGAGCCACACA AATAACTTAACTGTGCCTACACCATGTGTGGCTCCA TGGTGTAGGCACAGTTAAGTTATTTATAcctgtacatttgct TGCTCCGCACAGTG (SEQ ID NO: 145) ctgcctt (SEQ ID NO: 31) rs12475610 AAGTACCCCAAAGTGTGAGGGCCTTCCCTCTGCCG ACCGGACCGTTCTCGATGATGTGCGGCAGAGGGAA CACATCATCGAGAACGGTCCGGTCGCTcctgtacatttgctc GGCCCTCACAC (SEQ ID NO: 146) tgcctt (SEQ ID NO: 32) rs867983 GTGCTTCTGAAACTGTTATCTTCCCAGGAGCAATCT CACACTAATATGCTTTCTATCCCCAGATTGCTCCTG GGGGATAGAAAGCATATTAGTGTGGAGAcctgtacatttg GGAAGATAACAG (SEQ ID NO: 147) ctctgcctt (SEQ ID NO: 33) rs10207910 GGGCCAGTCTTTAAATGCTTCCTGGAAAATGTTACT ACTGCCAGAAGTTTATTTCATAGGTAGTAACATTTT ACCTATGAAATAAACTTCTGGCAGTggaccctgtacatttgct CCAGGAAGCATTTAA (SEQ ID NO: 148) ctgcctt (SEQ ID NO: 34) rs1990856 AAGGAAGCAGCGTGCAGTGCCATTCCTTCCTCCAC TGACTGGATCCTAACGAGGCTTACGTGGAGGAAGG GTAAGCCTCGTTAGGATCCAGTCAACTTcctgtacatttgct AATGGCACTGCAC (SEQ ID NO: 149) ctgcctt (SEQ ID NO: 35) rs73000450 ATTGGGGGTATACTGGAAAAGTATTTTTGGTGTTGA AAGGCTCGCTCACAGCCCAAACTTCAACACCAAAA AGTTTGGGCTGTGAGCGAGCCTTGTCAcctgtacatttgact ATACTTTTCCAG (SEQ ID NO: 150) gcctt (SEQ ID NO: 36) rs75059082 GGGATGTTTCTTGTCCTCGCTCAAGACAGAATTCGA TCCACCGCCACTGGCTCACTCTCGAATTCTGTCTTG GAGTGAGCCAGTGGCGGTGGAaggacctgtacatttgactgcct AGCGAGGAC (SEQ ID NO: 151) t (SEQ ID NO: 37) rs7648926 TTTGACACCAATAAAACGGAGTGCCACTGAAGGGT TCACTGGACTCTCCTCAGCTCAAAACCCTTCAGTGG TTTGAGCTGAGGAGAGTCCAGTGAACTAcctgtacatttgc CACTCCGTT (SEQ ID NO: 152) tagcctt (SEQ ID NO: 38) rs2306253 ACCGTACCTCTCCCCGACGTGGGCAGGCGTGAGTT GATTATGTTGTTCTCTAAACTGACAACTCACGCCTG GTCAGTTTAGAGAACAACATAATCGTAGcctgtacatttgc CCCACGTCGGGG (SEQ ID NO: 153) tagcctt (SEQ ID NO: 39) rs1316732 CTGCTTCTAGGGTTGGGATCTCCCAGGGAAGACCG GGCCTCGTTGCACATGGCAAGCCCGGTCTTCCCTGG GGCTTGCCATGTGCAACGAGGCCTTCTcctgtacatttgact GAGATCCCAAC (SEQ ID NO: 154) gcctt (SEQ ID NO: 40) rs2672761 GTCATTTTGCTGTTTGTTTTCTATATGCAGTATAAC GCTGATTACATATTAAGAGACAAAAATGTTATACT ATTTTTGTCTCTTAATATGTAATCAGCTGATcctgtacatt GCATATAGAAAACAAA (SEQ ID NO: 155) tgctagcctt (SEQ ID NO: 41) rs6882848 TAGGAGACAGAGAATGTTCTGTGGGACCACAACCG CCGGCATTAGAGCTCTTCTGTCTCGGTTGTGGTCCC AGACAGAAGAGCTCTAATGCCGGccGCcagtacatttgact ACAGAACATT (SEQ ID NO: 156) gcctt (SEQ ID NO: 42) rs1465127 CCTGTAACACACGCCCACAGGGGCTTCAGGAACTA TCCAAGAGAAAAGGGTGAATGTTTATAGTTCCTGA TAAACATTCACCCTTTTCTCTTGGAtATGcctgtacatttgct AGCCCCTGTGGGC (SEQ ID NO: 157) ctgcctt (SEQ ID NO: 43) rs1161899 TATTTGATAAATTAACCCTAGAACAACTATCTGCAC TGCGTTCCGTATGTGTTTCTGAGTGCAGATAGTTGT TCAGAAACACATACGGAACGCATTAGcctgtacatttgact TCTAG (SEQ ID NO: 158) gcctt (SEQ ID NO: 44) rs4615440 GAGCATCCTGAAGCAATTCTGTTTGTAATCCTGGAA TGAGTATAGGTTCCCAGTTACTACTTCCAGGATTAC GTAGTAACTGGGAACCTATACTCACCAGcctgtacatttgc AAACAGAATTGCT (SEQ ID NO: 159) tagcctt (SEQ ID NO: 45) rs9501710 TGAATTATTTTTCTTCCCCTTCATTTTTGTTTAAGCT TCGAATGGTACAAAAAACAATAGAGCTTAAACAAA CTATTGTTTTTTGTACCATTCGAgcCacctgtacatttgctagc AATGAAGGG (SEQ ID NO: 160)

ctt (SEQ ID NO: 46) rs6925983 AGAACAATGTCCACATGTTTCCTCTGTGCCATTACT CCGCCAATGGTAAGGGGACCATCTTAGTAATGGCA AAGATGGTCCCCTTACCATTGGCGGAAGCcctgtacattt CAGAGGAAACATGT (SEQ ID NO: 161) gctctgcctt (SEQ ID NO: 47) rs2972171 CATCCCACCCTGTCTCACTGGAGCCAGGATCCATG TCTGAAGCCTAGCTCACGGGACCTCATGGATCCTG AGGTCCCGTGAGCTAGGCTTCAGAAGGCcctgtacatttgc GCTCCAGTGAGACA (SEQ ID NO: 162) tctgcctt (SEQ ID NO: 48) rs62477557 TCCATCCTAAAGGACTTACAGTTTCTTAGAATAACA TCACTCCGAAAAATCACTCCATGTTATTCTAAGAAA TGGAGTGATTTTTCGGAGTGAcTGTcctgtacatttgctctgcct CTGTAAGTC (SEQ ID NO: 163) t (SEQ ID NO: 49) rs4876049 GAAAACAGTCAAAATGGCTGTCGACAATGAAATGG ATTGCCTGAGTCTCAACTGATGTATCCATTTCATTG ATACATCAGTTGAGACTCAGGCAATGTGAcctgtacattt TCGACAGCCA (SEQ ID NO: 164) gctctgcctt (SEQ ID NO: 50) rs1509186 GAAAGACTAATAATTTTGCCCATGATCACCTCACC AGAGCCGCCATTTCAGAGTGAGATGGTGAGGTGAT ATCTCACTCTGAAATGGCGGCTCTCAGGcctgtacatttgct CATGGGC (SEQ ID NO: 165) ctgcctt (SEQ ID NO: 51) rs1876904 AGTAGTCGGCATGGTGCTGAGCACCCTCCGGGAAC TCGGTAGCCTTTCAGGTAGGGACGGTTCCCGGAGG CGTCCCTACCTGAAAGGCTACCGAgccgcctgtacatttgctct GTGCTCAGCACCAT (SEQ ID NO: 166) gcctt (SEQ ID NO: 52) rs4880811 AATTCTAGCTCCAAAATCTGGGCTCCTGACCACAAT CACACTAATGCAAGGCACCTCTAACATTGTGGTCA GTTAGAGGTGCCTTGCATTAGTGTGGAGAcctgtacatttg GGAGCCCAGATTT (SEQ ID NO: 167) ctctgcctt (SEQ ID NO: 53) rs75196694 GAACCGTCACCAGGTCCTTTATTGCCTCTTCCAACA ATCTTGCGATGGATAATTTCTATTGTTGGAAGAGGC ATAGAAATTATCCATCGCAAGATCGTGcctgtacatttgctc AATAAAGGACCTG (SEQ ID NO: 168) tgcctt (SEQ ID NO: 54) rs2075545 AGTCCTAACCTAGGTTACAGCCCATCACAGCTGGA TGTATCAGGTTCTAAGCATCACCTGCTCCAGCTGTG GCAGGTGATGCTTAGAACCTGATACACCATcctgtacatt ATGGGCTGTAAC (SEQ ID NO: 169) tgctctgcctt (SEQ ID NO: 55) rs60326265 GTCAGGCTTAAGAGGCAGGGCCACCTAAACGTCTA CGCCAATACCCTGTGTTCTCAGTAGACGTTTAGGTG CTGAGAACACAGGGTATTGGCGagcccctgtacatttgctctgc GCCCTGCCT (SEQ ID NO: 170) ctt (SEQ ID NO: 56) rs953385 TTCAGATATGACTAGGGAATGTTTAGAAAGTACAC TCGCCTAGTCATGAAGCATGTGGCGTGTACTTTCTA GCCACATGCTTCATGACTAGGCGAggtccctgtacatttgctct AACATTCC (SEQ ID NO: 171) gcctt (SEQ ID NO: 57) rs77983336 CTCCAGGTATAGATGCAAGTAGGCTGGTAGATTTA GAACTGAACGAGTTTGTCTCCTCATTAAATCTACCA ATGAGGAGACAAACTCGTTCAGTTCAGCCcctgtacattt GCCTACTTGCA (SEQ ID NO: 172) gctctgcctt (SEQ ID NO: 58) rs1547149 CTGGGTGTAAAGTTTCTGTGCAAACCTTTGCTACAG CGGATGTCTTTGACTCGGCACGCACTGTAGCAAAG TGCGTGCCGAGTCAAAGACATCCGCGAGcctgtacatttgc GTTTGCACAGAAA (SEQ ID NO: 173) tctgcctt (SEQ ID NO: 59) rs3117978 GACCTGTAGTCACAAGTGTAGAGAGTTTGAGCTTC TCGTGGTGTGCCTTTCTAAGTCGAAGCTCAAACTCT GACTTAGAAAGGCACACCACGATAcTcctgtacatttgactg CTACACTTG (SEQ ID NO: 174) cat (SEQ ID NO: 60) rs9509962 TTCACTGGCGATCAACAGTAACCAATAAAATTCAC CCGGAAGGACTGTTGATTCATGAGTGAATTTTATTG TCATGAATCAACAGTCCTTCCGGTAGGcctgtacatttgctct GTTACTGTTGAT (SEQ ID NO: 175) gcctt (SEQ ID NO: 61) rs7139530 TCTCTGTAGTCAATTTGATTTTTATCAAGTTGCACT AGTGATAGGGTCTTAAAATATTTAGTGCAACTTGAT AAATATTTTAAGACCCTATCACTAGAGcctgtacatttgact AAAAATCAAA (SEQ ID NO: 176) gcctt (SEQ ID NO: 62) rs292476 CAGCCTGTGTTCAGGATCTCACAAAGTCTCTCATGA CATCGTATAGGTGCCCACAACTATTTTCATGAGAG AAATAGTTGTGGGCACCTATACGATGAATCcagtacatt ACTTTGTGAGATCCTG (SEQ ID NO: 177) tgctctgcctt (SEQ ID NO: 63) rs3000029 TTGTTCTCATCTCTCAGAAGCCCTTCTGTGGCCCAA GTCTGGCGATGGTCAATAATGTTTGGGCCACAGAA ACATTATTGACCATCGCCAGACTTGCcagtacatttgactg GGGCTTCTGA (SEQ ID NO: 178) cat (SEQ ID NO: 64) rs12434992 GAAGCCTAGGTATGTAAATTACAGGCTTGCAGAAG TCATGGCGAGCCACCTGCATTTACTTCTGCAAGCCT TAAATGCAGGTGGCTCGCCATGATGTCcagtacatttgctc GTAATTTAC (SEQ ID NO: 179) tgcctt (SEQ ID NO: 65) rs1760904 GGACGAGCCCCAGAAAAGTGGAAGAAGACTAATG AACTATCATCGGGGCCAAGGTGGCATCATTAGTCT ATGCCACCTTGGCCCCGATGATAGTTCCAGcctgtacatt TCTTCCACTTTTCTGG (SEQ ID NO: 180) tgctctgcctt (SEQ ID NO: 66) rs35567022 TGCCCTCGTCCCTACTGGTAAGAGGCATAAGGTGG TGGTGAGCACTTAGGCCCTTCCCCACCTTATGCCTC GGAAGGGCCTAAGTGCTCACCAGTCTcctgtacatttgctctg TTACCAGTAGGG (SEQ ID NO: 181) cctt (SEQ ID NO: 67) rs12910624 CTTAAAACTAAAACAGGAAAAAAAAATCAAAACC CACGTAACATCGTGATTACTGATTTGTTACGGTTTT GTAACAAATCAGTAATCACGATGTTACGTGGCTGcct GATTTTTTTTTC (SEQ ID NO: 182) gtacatttgctagcctt (SEQ ID NO: 68) rs34714665 AAATCAGTAAAATGTTTACAAGCAATATCTTTTACG GATGGTCGTCTTAGTTTTAAGATCGTAAAAGATATT ATCTTAAAACTAAGACGACCATCCTCAcctgtacatttgctc GCTTGTAA (SEQ ID NO: 183) tgcctt (SEQ ID NO: 69) rs6576457 CTACATAACAGAATTCAGTATGCAGTCATGATACA TGGCTCGATAACTTTGCTGAGAGTATGTATCATGAC TACTCTCAGCAAAGTTATCGAGCCAttGGcctgtacatttgct TGCATACTG (SEQ ID NO: 184) ctgcctt (SEQ ID NO: 70) rs2239669 TCCTCTCAGTCTCTGAGCTCTGTAGAGGAGCCTCAG CTTCTTCGGTTGCATCTGCCCCTGAGGCTCCTCTAC GGGCAGATGCAACCGAAGAAGGTATcctgtacatttgctctg AGAGCTCAG (SEQ ID NO: 185) cctt (SEQ ID NO: 71) rs1698232 ACACAAAACTAAAAGCACTTTTAATATTTCTTCAAA TCTCTCGTTGGAATTGAAAGAAGTTTTGAAGAAAT ACTTCTTTCAATTCCAACGAGAGAGCGAcctgtacatttgct ATTAAAAGTGC (SEQ ID NO: 186) ctgcctt (SEQ ID NO: 72) rs670962 AGCCACTCCACTCCTAGGTATCTGCCCGAGAGACA GACCGATGGTCCTTGTGCTTTCATGTCTCTCGGGCA TGAAAGCACAAGGACCATCGGTCGTCGcctgtacatttgct GATACCTAGGAG (SEQ ID NO: 187) ctgcctt (SEQ ID NO: 73) rs58445115 TGATCCCCAACAGAGAGAGGTACCCGGGATCTTCT GATGCGCACATGAACCACGTCAGAAGATCCCGGGT GACGTGGTTCATGTGCGCATCGTTTcctgtacatttgctctgcc ACCTCTCTCT (SEQ ID NO: 188) tt (SEQ ID NO: 74) rs59061318 TCCGAATTCTCCAACTTTCCTCCCAGCACGGGTCTG TGACTGAAGCCCGAGTCCCAAGGGCAGACCCGTGC CCCTTGGGACTCGGGCTTCAGTCATGTTcctgtacatttgct TGGGAGGAAAGTT (SEQ ID NO: 189) ctgcctt (SEQ ID NO: 75) rs6506015 TTTCCTTTCTTTCTTCCAAACTCCTCTTAATATTGGT TGGCGTTCTAGGAGGTCAAAATACCAATATTAAGA ATTTTGACCTCCTAGAACGCCAGGGAcctgtacatttgctctg GGAGTTTGGA (SEQ ID NO: 190) cctt (SEQ ID NO: 76) rs72634353 ACATAGAAGGTGTTCAGTAAATATTTCCTGACAGT TCCATACCTGCATTCATCAACTCCTACTGTCAGGAA AGGAGTTGATGAATGCAGGTATGGAgCAGcctgtacattt ATATTTACTGA (SEQ ID NO: 191) gctctgcctt (SEQ ID NO: 77) rs55677929 TCATGGCCGGTGGCCGGTTCTCACCCCTTTTGCTCC TGTTAGGCTCTGCGTGTCTGTTAGGAGCAAAAGGG TAACAGACACGCAGAGCCTAACAcTGCcctgtacatttgctc GTGAGAACCGGCCAC (SEQ ID NO: 192) tgcctt (SEQ ID NO: 78) rs6135141 GGAGATACTGACAATTGCAAGTTGGGCTGATATGC GGTTCTCGATATTTTCTGTTTTCATGCATATCAGCC ATGAAAACAGAAAATATCGAGAACCCGCAcctgtacattt CAACTTGCAAT (SEQ ID NO: 193) gctctgcctt (SEQ ID NO: 79) rs2050980 TAACAAAGACTAGCTTATACTACCCACACTTTCCTG TGGAACTACCGAAAAGAAAAATGACAGGAAAGTG TCATTTTTCTTTTCGGTAGTTCCAATTGcctgtacatttgctct TGGGTAGTATAA (SEQ ID NO: 194) gcctt (SEQ ID NO: 80) rs4815580 AACATTTTGTTTTATAATCTGCGTCTGATAATACCG CTCCATTGCAGAGTTTGTATATCGGTATTATCAGAC ATATACAAACTCTGCAATGGAGtTGGcctgtacatttgctctg GCAGA (SEQ ID NO: 195) cctt (SEQ ID NO: 81) rs463397 AGATGGTGAAGTAAAGATGAATAACATGAAGCAC AGAATCAAGGCCAATAGCATTCAAATGTGCTTCAT ATTTGAATGCTATTGGCCTTGATTCTCGTTcctgtacattt GTTATTCATCTT (SEQ ID NO: 196) gctctgcctt (SEQ ID NO: 82) rs7279689 TAGTGATATTTCAATACATATAATGTATAGTGATCA AATAATCGTACATTATGCTAATTACACTGATCACTA GTGTAATTAGCATAATGTACGATTATTAGACcctgtaca TACATTATATG (SEQ ID NO: 197) tttgctctgcctt (SEQ ID NO: 83) rs5748211 CTTTCTCTAGGTGCCGTACATGTTAGTGGGAGCTCC ACCTACCTCTCCAATCCAGGAAATAAGGAGCTCCC TTATTTCCTGGATTGGAGAGGTAGGTCTtGcctgtacattt ACTAACATGTACGG (SEQ ID NO: 198) gctctgcctt (SEQ ID NO: 84) rs79114187 AACTCTCAGTTTGGGCCACTGCTCTCCAGTTGCCTG TCACAGGTCGTAAGACTTAAAACTCCAGGCAACTG GAGTTTTAAGTCTTACGACCTGTGATAGGcctgtacatttg GAGAGCAGTGGCC (SEQ ID NO: 199) ctctgcctt (SEQ ID NO: 85) rs13164 ATGGCCAAGCCTTGGCTGTTGAGTAGGCACTGCCC TGGCTCAACCATACAGCACAACTGGGCAGTGCCTA AGTTGTGCTGTATGGTTGAGCCATGGtcctgtacatttgctct CTCAACAGCCAAG (SEQ ID NO: 200) gcctt (SEQ ID NO: 86) rs4633 TCCCGGGCTCCGCATGCTGCAGCACATGGTTCAGG AGTAAGGAATCAAGGAGCAGCGCATCCTGAACCAT ATGCGCTGCTCCTTGATTCCTTACTCCTAcctgtacatttgc GTGCTGCAGCATGCGGA (SEQ ID NO: 201) tctgcctt (SEQ ID NO: 87) rs13303106 GAAGGACCCCAGCTCCACCAACCAACAAAGGCACA atAAGGTGGGTGGGACGGACTGTGCCTTTGTTGGTT GTCCGTCCCACCCACCTTATTGcctgtacatttgctctgcctt GGTGGAGCT (SEQ ID NO: 202) (SEQ ID NO: 88) rs35273536 GAAATAGACCCTCGACAGACCCAAAGGGGCCCACG TACTTCTCTAACGTCACCACCGCATCACGTGGGCCC TGATGCGGTGGTGACGTTAGAGAAGTAAGGAcctgtac CTTTGGGTCTGTC (SEQ ID NO: 203) atttgctctgcctt (SEQ ID NO: 89) rs77129670 CCCAGATTTTGCTATTCCATACAGTTGACTGGACAT GTACGCCACCAAAATGAGTTCATGTCCAGTCAACT GAACTCATTTTGGTGGCGTACGGAGGcctgtacatttgctctg GTATGGAATA (SEQ ID NO: 204) cctt (SEQ ID NO: 90) rs17133064 ATTCTGAAAGGAATGAAAATGGGGTTTAAATGTCC GAAGATGCCCTCTGGACCTTAAGGACATTTAAACC TTAAGGTCCAGAGGGCATCTTCCAGTTcctgtacatttgctct CCATTTTC (SEQ ID NO: 205) gcctt (SEQ ID NO: 91) rs1161901 ATTCTGAAGATTTATCGTGAAAAAAAAAGAATGTA TGTTGGATTCGATATTAATAAAAGATTGTACATTCT CAATCTTTTATTAATATCGAATCCAACAtactcctgtacattt TTTTTTTTCAC (SEQ ID NO: 206) gctctgcctt (SEQ ID NO: 92) rs77474447 CAACCTGCCCCTCCCTGACCCGGGGCCCCCTTTCCT CAGGCGAGACTGGGCCCTGGAGGAAAGGGGGCCC CCAGGGCCCAGTCTCGCCTGCTGAAcctgtacatttgctctgc CGGGTCAGGGAG (SEQ ID NO: 207) ctt (SEQ ID NO: 93)

rs17756915 GTTGACTTCTTTTAAAATATGATCTTCACAATTACC CAGCTTCTCACAATTTGATTGGATGGTAATTGTGAA ATCCAATCAAATTGTGAGAAGCTGCGTTcctgtacatttgct GATCATATT (SEQ ID NO: 208) ctgcctt (SEQ ID NO: 94) rs341697 ATAGCTTTACCATTTTACCTTGCTCAATACGCACCC gGAGATATCACGTGTCTCTTTGTCTGGGGTGCGTAT CAGACAAAGAGACACGTGATATCTCCCAAcctgtacattt TGAGCAAGGTAA (SEQ ID NO: 209) gctctgcctt (SEQ ID NO: 95) rs10976019 TTTGTTAGCAGGGTTGGATCTAACCAGTGATGTGCG TGTAAGCTCACACTGACATGCCGCACATCACTGGTT GCATGTCAGTGTGAGCTTACATtTccctgtacatttgctctgcctt AGATCCAAC (SEQ ID NO: 210) (SEQ ID NO: 96) rs76408959 CCTCGTTACCTGCTTCTCATCTGTGATGCTCCCCAG CTTATACCTCGGCAAATGTTGCAGAGATCTGGGGA ATCTCTGCAACATTTGCCGAGGTATAAGcggAcctgtac GCATCACAGATGAGAAGC (SEQ ID NO: 211) atttgctctgcctt (SEQ ID NO: 97) rs9734804 GCCTGGGGCCGGGCGGCAGGGGCGCGCAGGGTGG CTACTAATAGAGGCCTCTGGGCCGCCACCCTGCGC CGGCCCAGAGGCCTCTATTAGTAGTAGACcctgtacattt GCCCCTGCCGCCCGG (SEQ ID NO: 212) gctctgcctt (SEQ ID NO: 98) rs12792188 GAGAGAGGGTGCTAGGCTGCTGGCCCAGCAAGGCC GTCCCTGCTTCCCTTGAGGCCTTGCTGGGCCAGCAG TCAAGGGAAGCAGGGACATTAGTcctgtacatttgctctgcctt CCTAG (SEQ ID NO: 213) (SEQ ID NO: 99) rs11611246 GGGGTTGGGGGGGTGGTGTTGAGGTATGTGTAAGT TAATTGGTGGCTATCATGAGCAATAGACTTACACA CTATTGCTCATGATAGCCACCAATTATCGAcctgtacattt TACCTCAACACCACCCC (SEQ ID NO: 214) gctctgcctt (SEQ ID NO: 100) rs79782920 AGGCGGGAACATAAACTAACAAAAAAGTATGTCAC GTACTGCCAGAAACATGTCACTGTGCTGTGACATA AGCACAGTGACATGTTTCTGGCAGTACATCTcctgtaca CTTTTTTGTTAGTTTATG (SEQ ID NO: 215) tttgctctgcctt (SEQ ID NO: 101) rs7989876 TGATGGGAGCACACCCCCCAATGACCCTGCCCCCA TATAAGGGCTTTTGCAGGTGTGGGGGCAGGGTCAT CACCTGCAAAAGCCCTTATAGACTcctgtacatttgctctgcctt TGGGGGGTGT (SEQ ID NO: 216) (SEQ ID NO: 102) rs7982082 TTAAAGCACATTAAAGCTCATTAGCCACTATGTCG GGATGGACTTGATTAGATAAGGCCTCGACATAGTG AGGCCTTATCTAATCAAGTCCATCCTGACcctgtacatttg GCTAATGAGC (SEQ ID NO: 217) ctctgcctt (SEQ ID NO: 103) rs77905703 TAGTATATCATATAAAAATAAAGACATCACCCAAC CACCTCGAAGGGTGATGTTTTTATGTTGGGTGATGT ATAAAAACATCACCCTTCGAGGTGTtggccctgtacatttgct CTT (SEQ ID NO: 218) ctgcctt (SEQ ID NO: 104) rs59329234 ATGTTGAACTCTTTTGTCAAAAGCCCCTTGTTGGAA GTCATGCGAAGTCTCTAGACTTTTCCAACAAGGGG AAGTCTAGAGACTTCGCATGACATCTTcctgtacatttgctct CTTTTGACA (SEQ ID NO: 219) gcctt (SEQ ID NO: 105) rs150926 AAACCGTATGTGATCTAGCAATGGAGGAGAGGGTC CTCTATGCAAGGTACTGGGGACTTCTGACCCTCTCC AGAAGTCCCCAGTACCTTGCATAGAGAaaatcctgtacattt TCCATTGCTAGAT (SEQ ID NO: 220) gctctgcctt (SEQ ID NO: 106) rs12450330 TCAAATTTCCCGTGATCATTACTGCCCATTTCCCAA GCTTAAGATGTGCAATGAGATATTTTGGGAAATGG AATATCTCATTGCACATCTTAAGCCACGGcctgtacatttg GCAGTAATGATCA (SEQ ID NO: 221) ctctgcctt (SEQ ID NO: 107) rs16948415 GGTCATGATAAGTAAGCAGTGAAACAAAGTAGACA AGGTAACGTTTTACTGATTCATATGTCTACTTTGTT TATGAATCAGTAAAACGTTACCTCTAGAcctgtacatttgct TCACTGCT (SEQ ID NO: 222) ctgcctt (SEQ ID NO: 108) rs11878153 CTTCACTCGCAGTAAATGTCTATTTCTCCTGTTTCAT GTGTGTAGTTAACTCAACCTTTTTAATGAAACAGGA TAAAAAGGTTGAGTTAACTACACACAAGGcctgtacattt GAAATAGACATTTAC (SEQ ID NO: 223) gctctgcctt (SEQ ID NO: 109) rs2279796 CTCTGCCCACGGTATACCTGGGAGAGTGCAGGTCC GACCGAACTCACCTTTCTGAAGGACCTGCACTCTCC TTCAGAAAGGTGAGTTCGGTCATCCTTcctgtacatttgctct CAGGTATACC (SEQ ID NO: 224) gcctt (SEQ ID NO: 110) rs6074167 TGATCATATGGTTTTTGTTTTTAATTCTGTTTATATG TTATCGGACTGTAAGTGTGATTCACCATATAAACA GTGAATCACACTTACAGTCCGATAACACCGcctgtacatt GAATTAAAAACAA (SEQ ID NO: 225) tgctctgcctt (SEQ ID NO: 111) rs2823170 AGATAGATGACTTAGAGGCCCTTGGGTGTAACAGA AGATGATTCTATTTGGGAAGACTGACTCTCTGTTAC GAGTCAGTCTTCCCAAATAGAATCATCTCATGAcctgt ACCCAAGGGCCT (SEQ ID NO: 226) acatttgctctgcctt (SEQ ID NO: 112) rs9984697 AATCTTCATAAAACCTCAGTGAATACTCTTTTTTCC TTATAATTCGTTATTATTAAGATTTTTTTAACAGGA TGTTAAAAAAATCTTAATAATAACGAATTATAACC AAAAAGAGTATTCACTGA (SEQ ID NO: 227) GGcctgtacatttgctctgcctt (SEQ ID NO: 113) rs17809319 CTTGCTTATGAACACTAATTTCATATATAAAACAAA GCAGTGGTTATCACAATAAATTTTTTGTTTTATATA AAATTTATTGTGATAACCACTGCATATGcctgtacatttgct TGAAATTAGT (SEQ ID NO: 228) ctgcctt (SEQ ID NO: 114)

TABLE-US-00002 TABLE 2 Exemplary Sinks SNP Sink Complement Sink Protector rs2246745 AAATAATCAGGAGAAGGAGAAGGCATGTTTGTTGG TGCTTCGAGCTCCTTGGAATCACCAACAAACATGC TGATTCCAAGGAGCTCGAAGCATAGG (SEQ ID NO: CTTCTCCT (SEQ ID NO: 343) 229) rs1805105 GGAGCAGCGTCTCTGCCATCGTCCTCATCCATGTCC TACACTCTTGGAAGTGTGACCAGGACATGGATGAG TGGTCACACTTCCAAGAGTGTATTTAGT (SEQ ID GACGATGGCAGAGA (SEQ ID NO: 344) NO: 230) rs3789806 AGGTAAATATTTACCACGTCTTGGTGTTTATTTTAC TATGATCTTGTATATAGACGGTAAAATAAACACCA CGTCTATATACAAGATCATACATTAC (SEQ ID NO: AGACGT (SEQ ID NO: 345) 231) rs9648696 CTTTCAGTCAGATGTATATGCATTTGGAATTGTTCT TCCGTTTCATCAATTCATACAGAACAATTCCAAATG GTATGAATTGATGAAACGGAGAAT (SEQ ID NO: CATATACA (SEQ ID NO: 346) 232) rs116952709 TATCTGCTAAGAAACAGGCATCCATATACAGAGAT GGTTATCAAAATCATCATTTTCATCTCTGTATATGG GAAAATGATGATTTTGATAACCTATA (SEQ ID NO: ATGCCTGT (SEQ ID NO: 347) 233) rs2511854 ATCTTGCCTTGCCTTCCACCGTAATACCAGCATAAT TGTGTATTAGTAGAAGCTTTAGTAGATTATGCTGGT CTACTAAAGCTTCTACTAATACACACATC (SEQ ID ATTACGGTGGAAGG (SEQ ID NO: 348) NO: 234) rs2510152 TGTCCAGTGATATGGTTATCATGTGAAACAAAACT TAGTCGAAAGTGACCCAGGTGAGTTTTGTTTCACAT CACCTGGGTCACTTTCGACTAGAAG (SEQ ID NO: GATAACCA (SEQ ID NO: 349) 235) rs2066827 ATTAAAGGCGCCGCCGGGCGGCTCCCGCTGACATC CGAAGCGCAGGAGAGCCAGGATGTCAGCGGGAGC CTGGCTCTCCTGCGCTTCGCGCGAATG (SEQ ID NO: CGCCCGGCG (SEQ ID NO: 350) 236) rs129974 GATTTGCGTTCTGCACTATGACATCATTTGGCCTTC TGCAATAAACAACTCACCCTGAAGGCCAAATGATG AGGGTGAGTTGTTTATTGCAGTAT (SEQ ID NO: 237) TCATAGTGCA (SEQ ID NO: 351) rs2228422 AGACTTCACCTTGTGATCTGCAGGGACTGACCTTG TCGGCCAACACAACAACACCAAGGTCAGTCCCTGC GTGTTGTTGTGTTGGCCGAAACC (SEQ ID NO: 238) AGATCACA (SEQ ID NO: 352) rs3738807 AGAGATCTCCAAAGACACTCCACGGAATGAGGGCT ACGCAAGACAAGGGCAACGAGCCCTCATTCCGTGG CGTTGCCCTTGTCTTGCGTGATT (SEQ ID NO: 239) AGTGTCT (SEQ ID NO: 353) rs2294976 CCTATACAATTGAGATGGTGGGGGAACCACAACAT TGTCCGCAGTCTGAATGAGTTATGTTGTGGTTCCCC AACTCATTCAGACTGCGGACAATTC (SEQ ID NO: CACCAT (SEQ ID NO: 354) 240) rs2305351 TTTTTATTGTATATGCATGCGCATCCCAAGGACCAA AAGCGGTGTAGCTGGTCTCTTGGTCCTTGGGATGCG GAGACCAGCTACACCGCTTAGGT (SEQ ID NO: 241) CATG (SEQ ID NO: 355) rs1630312 GCAGGCATCAAAGTGCACGACGTCCGGCTGAATGG TACATTGGCCAGCTGCGGAGCCATTCAGCCGGACG CTCCGCAGCTGGCCAATGTACTTAGT (SEQ ID NO: TCGTGCACTT (SEQ ID NO: 356) 242) rs10873531 TCTGCATTCCCTGTCACCGCGTCACTGGCCTTCAGA AGCAGGCACCTTGGCTCTGTCTGAAGGCCAGTGAC CAGAGCCAAGGTGCCTGCTTGTG (SEQ ID NO: 243) GCGGTGACAG (SEQ ID NO: 357) rs8005905 GCCCAAGTGTTTCTCTGGCATCTGATGGTGTCTGGA TCAAGCATAGTAGAGTGGTGGATCCAGACACCATC TCCACCACTCTACTATGCTTGAAATC (SEQ ID NO: AGATGCCAGAGAA (SEQ ID NO: 358) 244) rs117396186 GTTCAGATTTCACTGCCTCATGTTGATGTTTCTTTCC CATGTATTAAGACAGTCGTCATCTGGAAAGAAACA AGATGACGACTGTCTTAATACATGGTTC (SEQ ID TCAACATGAGGCA (SEQ ID NO: 359) NO: 245) rs34937835 TAGACACATTGTCATCATGGACGGGCGGTGGATAC GAAGACTCAGCTAGACGGCACGTATCCACCGCCCG GTGCCGTCTAGCTGAGTCTTCATAAT (SEQ ID NO: TCCATGATG (SEQ ID NO: 360) 246) rs17224367 GCTTGTCTTTGAAACTTCTTGGCAAGTCGGTTAAGA CAATATCCGTCGATTCCCAGATCTTAACCGACTTGC TCTGGGAATCGACGGATATTGGAAAA (SEQ ID NO: CAAGAAGTTT (SEQ ID NO: 361) 247) rs2303428 TACCTCCCATATTGGGGCCTACAAAACAAATTATA ATCATAATCTTGCTTTCTGATATAATTTGTTTTGTAG TCAGAAAGCAAGATTATGATCACAAA (SEQ ID NO: GCCCCA (SEQ ID NO: 362) 248) rs2229910 TGCCAATGACCACAGTGTCGGGCCCGGCATCCAGT GCCCACCACGCCCTCGTCACTGGATGCCGGGCCCG GACGAGGGCGTGGTGGGCTCGATT (SEQ ID NO: ACACTGTG (SEQ ID NO: 363) 249) rs200267496 ATCCCCCTTAAAATCACACTCACTTGCCGCGCATAG CAGCCAACATCGAGATGGCCTATGCGCGGCAAGTG GCCATCTCGATGTTGGCTGGACTA (SEQ ID NO: 250) AGTGTGAT (SEQ ID NO: 364) rs17334387 GGGGAGGTGAAGCTGTCCATCTCCTACAAAAACAA AGAATATGATGAAGAGTTTATTGTTTTTGTAGGAG TAAACTCTTCATCATATTCTCAACT (SEQ ID NO: ATGGACAGCTT (SEQ ID NO: 365) 251) rs706713 CGAGATTTTTTTCCTTCCAATATATTCTACGTAAGT TCGATCATAGGGACTTTCCGGGAACTTACGTAGAA TCCCGGAAAGTCCCTATGATCGAAATC (SEQ ID NO: TATATTGGAAG (SEQ ID NO: 366) 252) rs706714 CTTTTCCTTAAAAAGAAAAAGAAAGGGAGTCATTA CGTGTGATAAGTGGTTGCTTAATGACTCCCTTTCTT AGCAACCACTTATCACACGTATG (SEQ ID NO: 253) TTTC (SEQ ID NO: 367) rs290223 TGTGTGTTATGATTTCTCTTGCAGAGTTGTGAAAAC GAGCTCTTTTCACAGCCACGGTTTTCACAACTCTGC CGTGGCTGTGAAAAGAGCTCTGTA (SEQ ID NO: 254) AAGAGAA (SEQ ID NO: 368) rs1230345 GGCCCTGCAAATGCCCTCAGCAGAAGCCCCGTTGC CTTGCTTGCTCACTCCAGGAGGGCAACGGGGCTTCT CCTCCTGGAGTGAGCAAGCAAGAGTC (SEQ ID NO: GCTGAGGGCATTT (SEQ ID NO: 369) 255) rs16754 GCTACTCCAGGCACACGTCGCACATCCTGCAGGCA GCGTGACTTCCTCTTACTCTCTGCCTGCAGGATGTG GAGAGTAAGAGGAAGTCACGCAAAC (SEQ ID NO: CGACGTGTGC (SEQ ID NO: 370) 256) rs6667687 GATCTCTCCTGAGTCCTCACTAACAACAGGGGGTT GCTCTTGAAAACAATAAATCAACCCCCTGTTGTTAG GATTTATTGTTTTCAAGAGC (SEQ ID NO: 257) TGAGGAC (SEQ ID NO: 371) rs3737639 CCACTAGCCCTGGTTCAGGTCAGGGATGCCATGTT TATTTGCCTGGGCCCCGACAACATGGCATCCCTGAC GTCGGGGCCCAGGCAAATA (SEQ ID NO: 258) CTGAACCA (SEQ ID NO: 372) rs880724 TGGCAGCCTCACTGTGCGGAGCATGGAGCCACACG ACCACTGTGCCTACACCACGTGTGGCTCCATGCTCC TGGTGTAGGCACAGTGGT (SEQ ID NO: 259) GCACAGTG (SEQ ID NO: 373) rs12475610 AAGTACCCCAAAGTGTGAGGGCCTTCCCTCTGCCA AGCCATAAGTTCTCGATGATGTGTGGCAGAGGGAA CACATCATCGAGAACTTATGGCT (SEQ ID NO: 260) GGCCCTCACAC (SEQ ID NO: 374) rs867983 GTGCTTCTGAAACTGTTATCTTCCCAGGAGCAATTT ACTGATGCTTTCTATCCCCAAATTGCTCCTGGGAAG GGGGATAGAAAGCATCAGT (SEQ ID NO: 261) ATAACAG (SEQ ID NO: 375) rs10207910 GGGCCAGTCTTTAAATGCTTCCTGGAAAATGTTGCT ACAAGGTTTATTTCATAGGTAGCAACATTTTCCAGG ACCTATGAAATAAACCTTGT (SEQ ID NO: 262) AAGCATTTAA (SEQ ID NO: 376) rs1990856 AAGGAAGCAGCGTGCAGTGCCATTCCTTCCTCCAG ACCTAACCCTAACGAGGCTTACCTGGAGGAAGGAA GTAAGCCTCGTTAGGGTTAGGT (SEQ ID NO: 263) TGGCACTGCAC (SEQ ID NO: 377) rs73000450 ATTGGGGGTATATTGGAAAAGTATTTTTGGTGTTGA AGCTTGGCTCACAGCCCAAACTTCAACACCAAAAA AGTTTGGGCTGTGAGCCAAGCT (SEQ ID NO: 264) TACTTTTCCA (SEQ ID NO: 378) rs75059082 GGGATGTTTCTTGTCCTCGTTCAAGACAGAATTCGA GACTCCCCACTGGCTCACTCTCGAATTCTGTCTTGA GAGTGAGCCAGTGGGGAGTC (SEQ ID NO: 265) ACGAGGAC (SEQ ID NO: 379) rs7648926 TTTGACACCAATAAAATGGAGTGCCACTGAAGGGT TTCGTTCTCTCCTCAGCTCAAAACCCTTCAGTGGCA TTTGAGCTGAGGAGAGAACGAA (SEQ ID NO: 266) CTCCATT (SEQ ID NO: 380) rs2306253 ACCGTACCTCTGCCCGACGTGGGCAGGCGTGAGTT CTCTAATGTTCTCTAAACTGACAACTCACGCCTGCC GTCAGTTTAGAGAACATTAGAG (SEQ ID NO: 267) CACGTCGGGC (SEQ ID NO: 381) rs1316732 CTGCTTCTAGGGTTGGGAACTCCCAGGGAAGACCG TGTCGTGCACATGGCAAGCCCGGTCTTCCCTGGGA GGCTTGCCATGTGCACGACA (SEQ ID NO: 268) GTTCCCAAC (SEQ ID NO: 382) rs2672761 GTCATTTTGCTGTTTGTTTTCTATATGCGGTATAAC TCTCTCCTAAGAGACAAAAATGTTATACCGCATAT ATTTTTGTCTCTTAGGAGAGA (SEQ ID NO: 269) AGAAAACAAA (SEQ ID NO: 383) rs6882848 TAGGAGACAGAGAATGTTCTGTGGGACCACAACCA ACGGAGAGCTCTTCTGTCTTGGTTGTGGTCCCACAG AGACAGAAGAGCTCTCCGT (SEQ ID NO: 270) AACATT (SEQ ID NO: 384) rs1465127 CCTGTAACACACGCCCACAGGGGCTTCAGGAACTG CTGACTATAAAGGGTGAATGTTTACAGTTCCTGAA TAAACATTCACCCTTTATAGTCAG (SEQ ID NO: 271) GCCCCTGTGGGC (SEQ ID NO: 385) rs1161899 TATTTGATAAATTAACCCTAGAACAACTATCTGCGC GAGGATGGTATGTGTTTCTGAGCGCAGATAGTTGTT TCAGAAACACATACCATCCTC (SEQ ID NO: 272) CTAG (SEQ ID NO: 386) rs4615440 GAGCATCCTGAAGCAATTCTGTTTGTAATCCTGGG AATCTCATTCCCAGTTACTACTCCCAGGATTACAAA AGTAGTAACTGGGAATGAGATT (SEQ ID NO: 273) CAGAATTGCT (SEQ ID NO: 387) rs9501710 TGAATTATTTTTCTTCCCTTTCATTTTTGTTTAAGCT TCCATAACAAAAAACAATAGAGCTTAAACAAAAAT CTATTGTTTTTTGTTATGGA (SEQ ID NO: 274) GAAAGG (SEQ ID NO: 388) rs6925983 AGAACAATGTCCACATGTTTCCTCTGTGCCATTATT GTGATGGCAAGGGGACCATCTTAATAATGGCACAG AAGATGGTCCCCTTGCCATCAC (SEQ ID NO: 275) AGGAAACATGT (SEQ ID NO: 389) rs2972171 CATCCCACCCTGTCTCACTGGAGCCAGGATCCATA CAAGCTAGCTCACGGGACCTTATGGATCCTGGCTC AGGTCCCGTGAGCTAGCTTG (SEQ ID NO: 276) CAGTGAGACA (SEQ ID NO: 390) rs62477557 TCCATCCTAAAGGACTTACGGTTTCTTAGAATAACA TACTCTGAAAAATCACTCCATGTTATTCTAAGAAAC TGGAGTGATTTTTCAGAGTA (SEQ ID NO: 277) CGTAAGTC (SEQ ID NO: 391) rs4876049 GAAAACAGTCAAAATGGCTGTCAACAATGAAATGG ATGGTCTCTCAACTGATGTATCCATTTCATTGTTGA ATACATCAGTTGAGAGACCAT (SEQ ID NO: 278) CAGCCA (SEQ ID NO: 392) rs1509186 GAAAGACTAATAATTTTGCCCATGATCACCTCACT CTGTGCCATTTCAGAGTGAGATAGTGAGGTGATCA ATCTCACTCTGAAATGGCACAG (SEQ ID NO: 279) TGGGC (SEQ ID NO: 393) rs1876904 AGTAGTCGGCATGGTGCTGAGCATCCTCCGGGAAC TGCCTTTCAGGTAGGGACGGTTCCCGGAGGATGCT CGTCCCTACCTGAAAGGCA (SEQ ID NO: 280) CAGCACCAT (SEQ ID NO: 394) rs4880811 AATTCTAGCTCCAAAATCTGGGCTCCTGACCACAG CAATCGTAAAGGCACCTCTAACACTGTGGTCAGGA TGTTAGAGGTGCCTTTACGATTG (SEQ ID NO: 281) GCCCAGATTT (SEQ ID NO: 395) rs75196694 GAACCGTCACCAGGTCCTTTATTGCCTCTTCCAATA CACGGATGGATAATTTCTATTATTGGAAGAGGCAA ATAGAAATTATCCATCCGTG (SEQ ID NO: 282) TAAAGGACCTG (SEQ ID NO: 396) rs2075545 AGTCCTAACCTAGGTTACAGCCCATCACAGCTGGG ATTACATAACTAAGCATCACCTGCCCCAGCTGTGAT GCAGGTGATGCTTAGTTATGTAAT (SEQ ID NO: 283) GGGCTGTAAC (SEQ ID NO: 397) rs60326265 GTCAGGCTTAAGAGGCAGGGCCACCTAAACGTCTG AACCCTGTGTTCTCAGCAGACGTTTAGGTGGCCCTG CTGAGAACACAGGGTT (SEQ ID NO: 284) CCT (SEQ ID NO: 398) rs953385 TTCAGATATGACTAGGGAATGTTTAGAAAGTACAG

TACTACTCATGAAGCATGTGGCCTGTACTTTCTAAA GCCACATGCTTCATGAGTAGTA (SEQ ID NO: 285) CATTCC (SEQ ID NO: 399) rs77983336 CTCCAGGTATAGATGCAAGTAGGCTGGTAGATTTG TCCGAAGTTTGTCTCCTCATCAAATCTACCAGCCTA ATGAGGAGACAAACTTCGGA (SEQ ID NO: 286) CTTGCA (SEQ ID NO: 400) rs1547149 CTGGGTGTAAAGTTTCTGTGCAAACCTTTGCTACGG ACGCGTGACTCGGCACGCACCGTAGCAAAGGTTTG TGCGTGCCGAGTCACGCGT (SEQ ID NO: 287) CACAGAAA (SEQ ID NO: 401) rs3117978 GACCTGTAGTCACAAGTGTAGAGAGTTTGAGCTTT TAGACTAATGTGCCTTTCTAAGTCAAAGCTCAAACT GACTTAGAAAGGCACATTAGTCTA (SEQ ID NO: 288) CTCTACACTTG (SEQ ID NO: 402) rs9509962 TTCACTGGCGATCAACAGTAACTAATAAAATTCAC CTGCATGTACTGTTGATTCATGAGTGAATTTTATTA TCATGAATCAACAGTACATGCAG (SEQ ID NO: 289) GTTACTGTTGAT (SEQ ID NO: 403) rs7139530 TCTCTGTAGTCAATTTGATTTTTATCAAGTTGCATT CTACCGTCTTAAAATATTTAATGCAACTTGATAAAA AAATATTTTAAGACGGTAG (SEQ ID NO: 290) ATCAAA (SEQ ID NO: 404) rs292476 CAGCCTGTGTTCAGGATCTCACAGAGTCTCTCATGA TGGCTTATGCCCACAACTATTTTCATGAGAGACTCT AAATAGTTGTGGGCATAAGCCA (SEQ ID NO: 291) GTGAGATCCTG (SEQ ID NO: 405) rs3000029 TTGTTCTCATCTCTCAGATGCCCTTCTGTGGCCCAA TTAGGATATGGTCAATAATGTTTGGGCCACAGAAG ACATTATTGACCATATCCTAA (SEQ ID NO: 292) GGCATCTGA (SEQ ID NO: 406) rs12434992 GAAGCCTAGGTATGTAAATTATAGGCTTGCAGAAG AATCGAAAGCCACCTGCATTTACTTCTGCAAGCCTA TAAATGCAGGTGGCTTTCGATT (SEQ ID NO: 293) TAATTTAC (SEQ ID NO: 407) rs1760904 GGACGAGCCCCAGAAAAGTGGAAGAAGACTAATG AAGAGTATGGGGGCCAAGGTGGCACCATTAGTCTT GTGCCACCTTGGCCCCCATACTCTT (SEQ ID NO: CTTCCACTTTTCTGG (SEQ ID NO: 408) 294) rs35567022 TGCCCTCGGCCCTACTGGTAAGAGGCATAAGGTGG ATGAATAATTCACTTAGGCCCTTCCCCACCTTATGC GGAAGGGCCTAAGTGAATTATTCAT (SEQ ID NO: CTCTTACCAGTAGGG (SEQ ID NO: 409) 295) rs12910624 CTTAAAACTAAAACAGGAAAAAAAAATCAAAACC TGGATCCAATTACTGATTTGTTATGGTTTTGATTTTT ATAACAAATCAGTAATTGGATCCA (SEQ ID NO: 296) TTTTC (SEQ ID NO: 410) rs34714665 AAATCAGTAAAATGTTTACAAGCAATATCTTTTATG AGCGCTCTTAGTTTTAAGATCATAAAAGATATTGCT ATCTTAAAACTAAGAGCGCT (SEQ ID NO: 297) TGTAA (SEQ ID NO: 411) rs6576457 CTACATAACAGAATTCAGTATGCAGTCATGATACG GAACTCAACTTTGCTGAGAGTACGTATCATGACTG TACTCTCAGCAAAGTTGAGTTC (SEQ ID NO: 298) CATACTG (SEQ ID NO: 412) rs2239669 TCCTCTCAGTCTCTGAGCTCTGTAGAGGAGCCTCGG TCTAAGGTTGCATCTGCCCCCGAGGCTCCTCTACAG GGGCAGATGCAACCTTAGA (SEQ ID NO: 299) AGCTCAG (SEQ ID NO: 413) rs1698232 ACACAAAACTAAAAGCACTTTTAATATTTCTTCAG TCTCCGAATTGAAAGAAGTTCTGAAGAAATATTAA AACTTCTTTCAATTCGGAGA (SEQ ID NO: 300) AAGTGC (SEQ ID NO: 414) rs670962 AGCCACTCCACTCCTAGGTATCTGCCCAAGAGACA CCAGTGTCCTTGTGCTTTCATGTCTCTTGGGCAGAT TGAAAGCACAAGGACACTGG (SEQ ID NO: 301) ACCTAGGAG (SEQ ID NO: 415) rs58445115 TGATCCCCAACAGAGAGAGGTACCTGGGATCTTCT TGACACACATGAACCACGTCAGAAGATCCCAGGTA GACGTGGTTCATGTGTGTCA (SEQ ID NO: 302) CCTCTCTCT (SEQ ID NO: 416) rs59061318 TCCGAATTCTCCAACTTTCCTCCCAGCAAGGGTCTG TCATCGCCCGAGTCCCAAGGGCAGACCCTTGCTGG CCCTTGGGACTCGGGCGATGA (SEQ ID NO: 303) GAGGAAAGTT (SEQ ID NO: 417) rs6506015 TTTCCTTTCTTTCTTCCAAACTCCTGTTAATATTGGT CCACAGTTAGGAGGTCAAAATACCAATATTAACAG ATTTTGACCTCCTAACTGTGG (SEQ ID NO: 304) GAGTTTGGA (SEQ ID NO: 418) rs72634353 ACATAGAAGGTGTTCAGTAAATATTTCCTGACTGT ACGCACATTCATCAACTCCTACAGTCAGGAAATAT AGGAGTTGATGAATGTGCGT (SEQ ID NO: 305) TTACTGA (SEQ ID NO: 419) rs55677929 TCATGGCCGGTGGCCGGTTCTCACCCCTTTTGCTTC TCGCTCTGCGTGTCTGTTAGAAGCAAAAGGGGTGA TAACAGACACGCAGAGCGA (SEQ ID NO: 306) GAACCGGCCAC (SEQ ID NO: 420) rs6135141 GGAGATACTGACAATTGCAAGTTGGGCTGATATGT TGCCGTATTTTCTGTTTTCATACATATCAGCCCAAC ATGAAAACAGAAAATACGGCA (SEQ ID NO: 307) TTGCAAT (SEQ ID NO: 421) rs2050980 TAACAAAGACTAGCTTATACTACCCACGCTTTCCTG TGAACCGAAAAGAAAAATGACAGGAAAGCGTGGG TCATTTTTCTTTTCGGTTCA (SEQ ID NO: 308) TAGTATAA (SEQ ID NO: 422) rs4815580 AACATTTTGTTTTATAATCTGCGTCTGATAATACTG CTAGAGCAGAGTTTGTATATCAGTATTATCAGACG ATATACAAACTCTGCTCTAG (SEQ ID NO: 309) CAGA (SEQ ID NO: 423) rs463397 AGATGGTGAAGTAAAGATGAATAACATGAAGCAC TCAGAAGCCAATAGCATTCAAACGTGCTTCATGTT GTTTGAATGCTATTGGCTTCTGA (SEQ ID NO: 310) ATTCATCTT (SEQ ID NO: 424) rs7279689 TAGTGATATTTCAATACATATAATGTATAGTTATCA TGTACTTTATGCTAATTACACTGATAACTATACATT GTGTAATTAGCATAAAGTACA (SEQ ID NO: 311) ATATG (SEQ ID NO: 425) rs5748211 CTTTCTCTAGGTGCCGTACATGTTAGTGGGGGCTCC GAGCTTCAATCCAGGAAATAAGGAGCCCCCACTAA TTATTTCCTGGATTGAAGCTC (SEQ ID NO: 312) CATGTACGG (SEQ ID NO: 426) rs79114187 AACTCTCAGTTTGGGCCGCTGCTCTCCAGTTGCCTG GTCGTCTAAGACTTAAAACTCCAGGCAACTGGAGA GAGTTTTAAGTCTTAGACGAC (SEQ ID NO: 313) GCAGCGGCC (SEQ ID NO: 427) rs13164 ATGGCCAAGCCTTGGCTGTTGAGTAGGCAGTGCCC TCCAACCATACAGCACAACTGGGCACTGCCTACTC AGTTGTGCTGTATGGTTGGA (SEQ ID NO: 314) AACAGCCAAG (SEQ ID NO: 428) rs4633 TCCCGGGCTCCGCATGCTGCAGCACGTGGTTCAGG TGAATCAAGGAGCAGCGCATCCTGAACCACGTGCT ATGCGCTGCTCCTTGATTCA (SEQ ID NO: 315) GCAGCATGCGGA (SEQ ID NO: 429) rs13303106 GAAGGACCCCAGCTCCACCAACCAACAAAGGCAC GGTGGGTGGGACGGACCGTGCCTTTGTTGGTTGGT GGTCCGTCCCACCCACC (SEQ ID NO: 316) GGAGCT (SEQ ID NO: 430) rs35273536 GAAATAGACCCTCGACAGACCCAAAGGGGCCCAC TCTAACGTCACCACCGCATCATGTGGGCCCCTTTGG ATGATGCGGTGGTGACGTTAGA (SEQ ID NO: 317) GTCTGTC (SEQ ID NO: 431) rs77129670 CCCAGATTTTGCTAATCCATACAGTTGACTGGACAT gTGTCTCACCAAAATGAGTTCATGTCCAGTCAACTG GAACTCATTTTGGTGAGACACATA (SEQ ID NO: 318) TATGGATTA (SEQ ID NO: 432) rs17133064 ATTCTGAAAGGAATGAAAATGGGGTTTAAATGTCT gtATAGTTACCCCTCTGGACCTTAAAGACATTTAAA TTAAGGTCCAGAGGGGTAACTATACTT (SEQ ID NO: CCCCATTTTC (SEQ ID NO: 433) 319) rs1161901 ATTCTGAAGATTTATCATGAAAAAAAAAGAATGTA GACGGTTATTAATAAAAGATTGTACATTCTTTTTTT CAATCTTTTATTAATAACCGTC (SEQ ID NO: 320) TTCAT (SEQ ID NO: 434) rs77474447 CAACCTGCCCCTCCCTGACCCGGGGCCCCCTTTTCT TTCCATTCAAGACTGGGCCCTGGAGAAAAGGGGGC CCAGGGCCCAGTCTTGAATGGAA (SEQ ID NO: 321) CCCGGGTCAGGGAG (SEQ ID NO: 435) rs17756915 GTTGACTTCTTTTAAAATATGATCTTCACAATTATC caTCGTCTAACAATTTGATTGGATGATAATTGTGAA ATCCAATCAAATTGTTAGACGATGCT (SEQ ID NO: GATCATATT (SEQ ID NO: 436) 322) rs341697 ATAGCTTTACCATTTTACCTTGCTCAATACACACCC GAGTGTGTCTCTTTGTCTGGGGTGTGTATTGAGCAA CAGACAAAGAGACACACTCA (SEQ ID NO: 323) GGTAA (SEQ ID NO: 437) rs10976019 TTTGTTAGCAGGGTTGGATCTAACCAGTGATGTGTG AAGGATTCACACTGACATGCCACACATCACTGGTT GCATGTCAGTGTGAATCCTT (SEQ ID NO: 324) AGATCCAAC (SEQ ID NO: 438) rs76408959 CCTCGTTACCTGCTTCTCATCTGTGATGCTCCCCTG TCATTATGAATGTTGCAGAGATCAGGGGAGCATCA ATCTCTGCAACATTCATAATGA (SEQ ID NO: 325) CAGATGAGAAGC (SEQ ID NO: 439) rs9734804 GCCTGGGGCCGGGCGGCAGGGGCGCGCAGGGTGG gTCTAAAGAGGCCTCTGGGCCCCCACCCTGCGCGCC GGGCCCAGAGGCCTCTTTAGACATG (SEQ ID NO: CCTGCCGCCCGG (SEQ ID NO: 440) 326) rs12792188 GAGAGAGGGTGCTAGGCTGCTGGCCCAGCAAGGC CCCTGCTTCCCTTGACGCCTTGCTGGGCCAGCAGCC GTCAAGGGAAGCAGGGA (SEQ ID NO: 327) TAG (SEQ ID NO: 441) rs11611246 GGGGTTGGGGGGGTGGTGTTGAGGTATGTGTAAGG GGCGATATCATGAGCAATAGCCTTACACATACCTC CTATTGCTCATGATATCGCCG (SEQ ID NO: 328) AACACCACCCC (SEQ ID NO: 442) rs79782920 AGGCGGGAACATAAACTAACAAAAAAGTATGTCAT tTGCTCAACATGTCACTGTGCTATGACATACTTTTTT AGCACAGTGACATGTTGAGCAACCT (SEQ ID NO: GTTAGTTTATG (SEQ ID NO: 443) 329) rs7989876 TGATGGGAGCACACCCCCCAATGACCCTGCCCCCG AGGGCTTTTGCAGGTGCGGGGGCAGGGTCATTGGG CACCTGCAAAAGCCCT (SEQ ID NO: 330) GGGTGT (SEQ ID NO: 444) rs7982082 TTAAAGCACATTAAAGCTCATTAGCCACTATGTCA TGAATCGATTAGATAAGGCCTTGACATAGTGGCTA AGGCCTTATCTAATCGATTCA (SEQ ID NO: 331) ATGAGC (SEQ ID NO: 445) rs77905703 TAGTATATCATATAAAAATAAAGACATCACCCAAT gTCCGAGGGTGATGTTTTTATATTGGGTGATGTCTT ATAAAAACATCACCCTCGGACTAA (SEQ ID NO: 332) (SEQ ID NO: 446) rs59329234 ATGTTGAACTCTTTTGTCAAAAGCCCCTTGTTGGGA AGACCATTAAGTCTCTAGACTTTCCCAACAAGGGG AAGTCTAGAGACTTAATGGTCT (SEQ ID NO: 333) CTTTTGACA (SEQ ID NO: 447) rs150926 AAACCGTATGTGATCTAGCAATGGAGGAGAGGGTG taTGCTCGTACTGGGGACTTCTCACCCTCTCCTCCAT AGAAGTCCCCAGTACGAGCATAAC (SEQ ID NO: TGCTAGAT (SEQ ID NO: 448) 334) rs12450330 TCAAATTTCCCGTGATCGTTACTGCCCATTTCCCAA cCAACCATGCAATGAGATATTTTGGGAAATGGGCA AATATCTCATTGCATGGTTGGGTT (SEQ ID NO: 335) GTAACGATCA (SEQ ID NO: 449) rs16948415 GGTCATGATAAGTAAGCAGTGAAACAAAGTAGAC CGATTTTTTACTGATTCATACGTCTACTTTGTTTCAC GTATGAATCAGTAAAAAATCGA (SEQ ID NO: 336) TGCT (SEQ ID NO: 450) rs11878153 CTTCACTCGCAGTAAATGTCTATTTCTCCTGTTTTAT AGTGAGACTATCTCAACCTTTTTAATAAAACAGGA TAAAAAGGTTGAGATAGTCTCACT (SEQ ID NO: 337) GAAATAGACATTTAC (SEQ ID NO: 451) rs2279796 CTCTGCCCACGGTATACCTGGGAGAGTGCAGGTCT TAATAATAGACTCACCTTTCTGAAAGACCTGCACTC TTCAGAAAGGTGAGTCTATTATTAC (SEQ ID NO: TCCCAGGTATACC (SEQ ID NO: 452) 338) rs6074167 TGATCATATGGTTTTTGTTTTTAATTCTGTTTATGTG taTTCGGTGAAGTGTGATTCACCACATAAACAGAAT GTGAATCACACTTCACCGAATAAG (SEQ ID NO: 339) TAAAAACAA (SEQ ID NO: 453) rs2823170 AGATAGATGACTTAGAGGCCCTTGGGTGTAACAGT GAAGAGTGGGAAGACTGACTCACTGTTACACCCAA GAGTCAGTCTTCCCACTCTTCAT (SEQ ID NO: 340) GGGCCT (SEQ ID NO: 454) rs9984697 AATCTTCATAAAACCTCAGTGAATACTCTTTTTTAC ACTATAATAGATTACTAGATTTTTTTAACAGTAAAA TGTTAAAAAAATCTAGTAATCTATTATAGT (SEQ ID AAGAGTATTCACTGA (SEQ ID NO: 455) NO: 341) rs17809319 CTTGCTTATGAACACTAATTTCATATATAAAACAGA taTGATCCATCACAATAAATTTTCTGTTTTATATATG AAATTTATTGTGATGGATCATATA (SEQ ID NO: 342) AAATTAGT (SEQ ID NO: 456)

TABLE-US-00003 TABLE 3 Exemplary PCR Primers SNP fPseq with adaptor rPseq with adaptor rs2246745 tcgtcggcagcgtcagatgtgtataagagacagCCAAGCACATGGAT gtctcgtgggctcggagatgtgtataagagacagGAGACAGGAAAGG CAGTGTT (SEQ ID NO: 457) GAAGGAGT (SEQ ID NO: 571) rs1805105 tcgtcggcagcgtcagatgtgtataagagacagAGGGAAGGGCATAT gtctcgtgggctcggagatgtgtataagagacagTGTCTCCAGGAGCA CTGGATAC (SEQ ID NO: 458) GCTTC (SEQ ID NO: 572) rs3789806 tcgtcggcagcgtcagatgtgtataagagacagTTCACGCTTACCCAG gtctcgtgggctcggagatgtgtataagagacagATCAACAACAGGGA GAGTT (SEQ ID NO: 459) CCAGGTA (SEQ ID NO: 573) rs9648696 tcgtcggcagcgtcagatgtgtataagagacagAAGGTAACTGTCCA gtctcgtgggctcggagatgtgtataagagacagTGTTCTAACAGGCA GTCATCAATTC (SEQ ID NO: 460) CCAGAAGT (SEQ ID NO: 574) rs116952709 tcgtcggcagcgtcagatgtgtataagagacagGCTGTGTAGTTTCTA gtctcgtgggctcggagatgtgtataagagacagACCACTCTGGCTGC AGGGTCG (SEQ ID NO: 461) AAAGT (SEQ ID NO: 575) rs2511854 tcgtcggcagcgtcagatgtgtataagagacagCCCAGACGAGTACA gtctcgtgggctcggagatgtgtataagagacagAAGTTATTGTTATTC GCTCA (SEQ ID NO: 462) TTGATGGTTCTTTTGA (SEQ ID NO: 576) rs2510152 tcgtcggcagcgtcagatgtgtataagagacagAGAATCCTGATCTGA gtctcgtgggctcggagatgtgtataagagacagGTTCCAATGAATTC CTGGCTT (SEQ ID NO: 463) AATTATGCTGTCA (SEQ ID NO: 577) rs2066827 tcgtcggcagcgtcagatgtgtataagagacagCTTGCCCGAGTTCTA gtctcgtgggctcggagatgtgtataagagacagCAAATGCGTGTCCT CTACAGA (SEQ ID NO: 464) CAGAGTT (SEQ ID NO: 578) rs129974 tcgtcggcagcgtcagatgtgtataagagacagCTGGCTCTGTGCAGA gtctcgtgggctcggagatgtgtataagagacagTCCTAGTTTCGTTGA ACTG (SEQ ID NO: 465) TTGCAAGG (SEQ ID NO: 579) rs2228422 tcgtcggcagcgtcagatgtgtataagagacagGCCCAGATCGTGTGC gtctcgtgggctcggagatgtgtataagagacagTCCACCATGGGAAA TC (SEQ ID NO: 466) CCTGG (SEQ ID NO: 580) rs3738807 tcgtcggcagcgtcagatgtgtataagagacagGCTGGACTGGCTTCA gtctcgtgggctcggagatgtgtataagagacagTTCACAGGGGCATG CAA (SEQ ID NO: 467) TTTTAGC (SEQ ID NO: 581) rs2294976 tcgtcggcagcgtcagatgtgtataagagacagCTCCTCGTGGATCCA gtctcgtgggctcggagatgtgtataagagacagAAAGGCAAAGAGG AAATTGC (SEQ ID NO: 468) GCTTTGG (SEQ ID NO: 582) rs2305351 tcgtcggcagcgtcagatgtgtataagagacagGGTTTCAAGCCCTCT gtctcgtgggctcggagatgtgtataagagacagCTGATCTATGATTCT GCA (SEQ ID NO: 469) AAATTTTGCTGTCA (SEQ ID NO: 583) rs1630312 tcgtcggcagcgtcagatgtgtataagagacagCAGCCCAAGCCATTG gtctcgtgggctcggagatgtgtataagagacagAACCTTGGAGATAA TCT (SEQ ID NO: 470) CTCTGAAGGA (SEQ ID NO: 584) rs10873531 tcgtcggcagcgtcagatgtgtataagagacagAGCCTAAGCAATAT gtctcgtgggctcggagatgtgtataagagacagGTCTCTGGAAACAG AAATGGCTGC (SEQ ID NO: 471) CCCTTC (SEQ ID NO: 585) rs8005905 tcgtcggcagcgtcagatgtgtataagagacagCAGAGTAGAGTGGT gtctcgtgggctcggagatgtgtataagagacagAGATTGTGTTTATG GGATCCA (SEQ ID NO: 472) TTCCCAGCA (SEQ ID NO: 586) rs117396186 tcgtcggcagcgtcagatgtgtataagagacagTGAACACAGCCCAC gtctcgtgggctcggagatgtgtataagagacagAACAACAACAACAG CTCA (SEQ ID NO: 473) AAACCAGTTAG (SEQ ID NO: 587) rs34937835 tcgtcggcagcgtcagatgtgtataagagacagCTCTGCACTCCATGC gtctcgtgggctcggagatgtgtataagagacagGCACCTTTCACAAT CAAC (SEQ ID NO: 474) GGTTAAGG (SEQ ID NO: 588) rs17224367 tcgtcggcagcgtcagatgtgtataagagacagTGGAAGCTTTTGTAG gtctcgtgggctcggagatgtgtataagagacagTGATAGAGTCGGTA AAGATGCA (SEQ ID NO: 475) ACAATCTTGTAAG (SEQ ID NO: 589) rs2303428 tcgtcggcagcgtcagatgtgtataagagacagCAGTGTACAGTTTAG gtctcgtgggctcggagatgtgtataagagacagCCCAATTTGGGCCA GACTAACAATCC (SEQ ID NO: 476) TGAGT (SEQ ID NO: 590) rs2229910 tcgtcggcagcgtcagatgtgtataagagacagGTCATCAGTGGTGAG gtctcgtgggctcggagatgtgtataagagacagCAGTTGTGTCCCTG GAGGA (SEQ ID NO: 477) ACGG (SEQ ID NO: 591) rs200267496 tcgtcggcagcgtcagatgtgtataagagacagCCAAGCTACATCAGT gtctcgtgggctcggagatgtgtataagagacagGTTTCCTTTTACTCC GATGTGG (SEQ ID NO: 478) CTAGAGGTT (SEQ ID NO: 592) rs17334387 tcgtcggcagcgtcagatgtgtataagagacagTGTGCAGGCACTTAC gtctcgtgggctcggagatgtgtataagagacagTCATGGTTTCATTTG CAAG (SEQ ID NO: 479) TCCCTACA (SEQ ID NO: 593) rs706713 tcgtcggcagcgtcagatgtgtataagagacagGAAGCCAGGCCTGA gtctcgtgggctcggagatgtgtataagagacagAGGAAGAGGCCGA AGAAA (SEQ ID NO: 480) GGTG (SEQ ID NO: 594) rs706714 tcgtcggcagcgtcagatgtgtataagagacagACTGAAGCAGATGTT gtctcgtgggctcggagatgtgtataagagacagCCCAGAACATAACG GAACAACA (SEQ ID NO: 481) ACTCAACC (SEQ ID NO: 595) rs290223 tcgtcggcagcgtcagatgtgtataagagacagTCATTGGCCTCGTTT gtctcgtgggctcggagatgtgtataagagacagCACAGGGGGATTAT TTCAGT (SEQ ID NO: 482) GCTTCAC (SEQ ID NO: 596) rs1230345 tcgtcggcagcgtcagatgtgtataagagacagCCTGGTTGCTTGGCA gtctcgtgggctcggagatgtgtataagagacagTGAAGGAAGGCCTG CA (SEQ ID NO: 483) GAGAA (SEQ ID NO: 597) rs16754 tcgtcggcagcgtcagatgtgtataagagacagCTCCCTCAAGACCTA gtctcgtgggctcggagatgtgtataagagacagCGTTTCTCACTGGTC CGTGA (SEQ ID NO: 484) TCAGATG (SEQ ID NO: 598) rs6667687 tcgtcggcagcgtcagatgtgtataagagacaggttaaagacggcacttccaacag gtctcgtgggctcggagatgtgtataagagacagtgacccttgccctggtaga (SEQ ID NO: 485) (SEQ ID NO: 599) rs3737639 tcgtcggcagcgtcagatgtgtataagagacagtcctccgtggctctccc (SEQ gtctcgtgggctcggagatgtgtataagagacagctgccctggagccactag ID NO: 486) (SEQ ID NO: 600) rs880724 tcgtcggcagcgtcagatgtgtataagagacagagcttggggacacctctga gtctcgtgggctcggagatgtgtataagagacagaccacgaacagcagaagca (SEQ ID NO: 487) (SEQ ID NO: 601) rs12475610 tcgtcggcagcgtcagatgtgtataagagacaggatggttccagctgcgct (SEQ gtctcgtgggctcggagatgtgtataagagacagtgtgtatcatcatctctaatttaaag ID NO: 488) aaaaagtac (SEQ ID NO: 602) rs867983 tcgtcggcagcgtcagatgtgtataagagacagtccgatataagttaacaatgcaatg gtctcgtgggctcggagatgtgtataagagacagtggccagccaagggga (SEQ tca (SEQ ID NO: 489) ID NO: 603) rs10207910 tcgtcggcagcgtcagatgtgtataagagacagtgatcttatttatatattttcagtcattt gtctcgtgggctcggagatgtgtataagagacagccgtgtgctccatcttacaatac gtcctac (SEQ ID NO: 490) (SEQ ID NO: 604) rs1990856 tcgtcggcagcgtcagatgtgtataagagacaggctccaacatttcatccaggatttg gtctcgtgggctcggagatgtgtataagagacagggcccagcgtgtgtatga (SEQ ID NO: 491) (SEQ ID NO: 605) rs73000450 tcgtcggcagcgtcagatgtgtataagagacaggccattacacctaagcaccatcta gtctcgtgggctcggagatgtgtataagagacagtctccatttgtagctgaattcttgtc c (SEQ ID NO: 492) (SEQ ID NO: 606) rs75059082 tcgtcggcagcgtcagatgtgtataagagacagaaagctaaagcagagaatgaagt gtctcgtgggctcggagatgtgtataagagacagtgtttttgtttttttaccactggctc tga (SEQ ID NO: 493) (SEQ ID NO: 607) rs7648926 tcgtcggcagcgtcagatgtgtataagagacagaatatcatgtcctatttctcctcagct gtctcgtgggctcggagatgtgtataagagacaggccaaacagtgttttgtagaccat (SEQ ID NO: 494) t (SEQ ID NO: 608) rs2306253 tcgtcggcagcgtcagatgtgtataagagacagggagctgtgacaatgaaaatgca gtctcgtgggctcggagatgtgtataagagacaggatcagggggcagaaggatg g (SEQ ID NO: 495) (SEQ ID NO: 609) rs1316732 tcgtcggcagcgtcagatgtgtataagagacagccgtcaccgtggagtttcc gtctcgtgggctcggagatgtgtataagagacagccctgctctgacaccagg (SEQ ID NO: 496) (SEQ ID NO: 610) rs2672761 tcgtcggcagcgtcagatgtgtataagagacagtggaagagcttacatttaagtgatt gtctcgtgggctcggagatgtgtataagagacagtgatactaccaaaataatcaaaa actg (SEQ ID NO: 497) gcacaaa (SEQ ID NO: 611) rs6882848 tcgtcggcagcgtcagatgtgtataagagacagaaagtggtggtttttaaccccttc gtctcgtgggctcggagatgtgtataagagacagtccttggcagccgttcc (SEQ (SEQ ID NO: 498) ID NO: 612) rs1465127 tcgtcggcagcgtcagatgtgtataagagacaggcctatagatggcaaattaagaga gtctcgtgggctcggagatgtgtataagagacagaacacacagacaggcaggtt gca (SEQ ID NO: 499) (SEQ ID NO: 613) rs1161899 tcgtcggcagcgtcagatgtgtataagagacagaaaaagtgaatcaatagagtacta gtctcgtgggctcggagatgtgtataagagacagagtgctcaatagttaccataatgc gtgcta (SEQ ID NO: 500) tatattg (SEQ ID NO: 614) rs4615440 tcgtcggcagcgtcagatgtgtataagagacagatgggaagggtacgatgttacc gtctcgtgggctcggagatgtgtataagagacagcctcctctctgtgtccatagaac (SEQ ID NO: 501) (SEQ ID NO: 615) rs9501710 tcgtcggcagcgtcagatgtgtataagagacaggattaggataattttccagctcaaa gtctcgtgggctcggagatgtgtataagagacagtcaatggttttaccatttaaaaattc gaaaat (SEQ ID NO: 502) cctatc (SEQ ID NO: 616) rs6925983 tcgtcggcagcgtcagatgtgtataagagacagcctctaaaactagagtgcctatag gtctcgtgggctcggagatgtgtataagagacagctcagttgctcagaacaatgtcc aatttattg (SEQ ID NO: 503) (SEQ ID NO: 617) rs2972171 tcgtcggcagcgtcagatgtgtataagagacagagtatttagttaacggttgttttacg gtctcgtgggctcggagatgtgtataagagacagggagtttcatcaccaagtccaca ct (SEQ ID NO: 504) (SEQ ID NO: 618) rs62477557 tcgtcggcagcgtcagatgtgtataagagacaggaattttgatgaaaacattcctgcta gtctcgtgggctcggagatgtgtataagagacagcccttgctatcaatattcaaagag tca (SEQ ID NO: 505) agaaa (SEQ ID NO: 619) rs4876049 tcgtcggcagcgtcagatgtgtataagagacagcacagtgttctacggtatacaagta gtctcgtgggctcggagatgtgtataagagacaggctcgtaggtgtgcaccat tct (SEQ ID NO: 506) (SEQ ID NO: 620) rs1509186 tcgtcggcagcgtcagatgtgtataagagacaggctaccttatagtcttccctagctta gtctcgtgggctcggagatgtgtataagagacagagaacattcaatgatataaaagg ataattt (SEQ ID NO: 507) aataagagaac (SEQ ID NO: 621) rs1876904 tcgtcggcagcgtcagatgtgtataagagacaggcagggtggctgcgt (SEQ gtctcgtgggctcggagatgtgtataagagacagtccttggagctgacatggc ID NO: 508) (SEQ ID NO: 622) rs4880811 tcgtcggcagcgtcagatgtgtataagagacaggcttggaatgaaatccctatcccta gtctcgtgggctcggagatgtgtataagagacaggggatctctcatctcaggcttg t (SEQ ID NO: 509) (SEQ ID NO: 623) rs75196694 tcgtcggcagcgtcagatgtgtataagagacagcagtggtcctgacgttcgg gtctcgtgggctcggagatgtgtataagagacagtgtgtgccctcgaaccg (SEQ (SEQ ID NO: 510) ID NO: 624) rs2075545 tcgtcggcagcgtcagatgtgtataagagacagcctaatacattaaagcagtcactttt gtctcgtgggctcggagatgtgtataagagacagcgaccccatctctgagtcct cct (SEQ ID NO: 511) (SEQ ID NO: 625) rs60326265 tcgtcggcagcgtcagatgtgtataagagacagggctcacgtcatgggca (SEQ gtctcgtgggctcggagatgtgtataagagacagctaggagcagtcaggcttaaga ID NO: 512) g (SEQ ID NO: 626) rs953385 tcgtcggcagcgtcagatgtgtataagagacagagaactcaaacaagatttaaggtc gtctcgtgggctcggagatgtgtataagagacagtgaagaacatgcttgccatagc tagaaa (SEQ ID NO: 513) (SEQ ID NO: 627) rs77983336 tcgtcggcagcgtcagatgtgtataagagacagcctccactcaaagtttctggc gtctcgtgggctcggagatgtgtataagagacaggcactattcaggcaaaggctc (SEQ ID NO: 514) (SEQ ID NO: 628) rs1547149 tcgtcggcagcgtcagatgtgtataagagacagtggcacagactttattggctct gtctcgtgggctcggagatgtgtataagagacagcccagaggattaagagacatgg (SEQ ID NO: 515) c (SEQ ID NO: 629) rs3117978 tcgtcggcagcgtcagatgtgtataagagacagcagagatcatttctattgccacagg gtctcgtgggctcggagatgtgtataagagacagaagctctagaaaaggcaaaact (SEQ ID NO: 516) aaacta (SEQ ID NO: 630) rs9509962 tcgtcggcagcgtcagatgtgtataagagacaggtaagcctagtgcccagtatatcat gtctcgtgggctcggagatgtgtataagagacagtctcctattcagcctataagtgttt (SEQ ID NO: 517) ctaa (SEQ ID NO: 631) rs7139530 tcgtcggcagcgtcagatgtgtataagagacaggctagtgtacgatatgtgtgtattg

gtctcgtgggctcggagatgtgtataagagacagaaaacgacttacacatacctaaa attaa (SEQ ID NO: 518) atgaaattt (SEQ ID NO: 632) rs292476 tcgtcggcagcgtcagatgtgtataagagacagaccctcctgcttatgtggttac gtctcgtgggctcggagatgtgtataagagacagtttgatttgggagcaaagaatga (SEQ ID NO: 519) gt (SEQ ID NO: 633) rs3000029 tcgtcggcagcgtcagatgtgtataagagacagccctgggtcacacacaaca gtctcgtgggctcggagatgtgtataagagacaggcatctctatgccaaactggtcat (SEQ ID NO: 520) a (SEQ ID NO: 634) rs12434992 tcgtcggcagcgtcagatgtgtataagagacagaagtgagtgggaacagtcatattg gtctcgtgggctcggagatgtgtataagagacagccaaactacttcttttctaacagaa a (SEQ ID NO: 521) agca (SEQ ID NO: 635) rs1760904 tcgtcggcagcgtcagatgtgtataagagacaggcagataggtacagaggcgtct gtctcgtgggctcggagatgtgtataagagacaggcatctcttgtgtcagccct (SEQ ID NO: 522) (SEQ ID NO: 636) rs35567022 tcgtcggcagcgtcagatgtgtataagagacagttgactttccagaccccactta gtctcgtgggctcggagatgtgtataagagacagccagggaaaaaatatgttcgatg (SEQ ID NO: 523) cc (SEQ ID NO: 637) rs12910624 tcgtcggcagcgtcagatgtgtataagagacaggacttgggaagtttttgattactaat gtctcgtgggctcggagatgtgtataagagacaggataacatagtaatgaatacattt tcaat (SEQ ID NO: 524) ctaaaaccgtaa (SEQ ID NO: 638) rs34714665 tcgtcggcagcgtcagatgtgtataagagacagctttttatatattgcacactctaaaaa gtctcgtgggctcggagatgtgtataagagacagtccagaagattagttgaaaatttg gaggt (SEQ ID NO: 525) agtacaa (SEQ ID NO: 639) rs6576457 tcgtcggcagcgtcagatgtgtataagagacaggggaaaaacaaaattgtctcaaaa gtctcgtgggctcggagatgtgtataagagacagcgttttgtcatttttgcagataaatg aatgt (SEQ ID NO: 526) tagt (SEQ ID NO: 640) rs2239669 tcgtcggcagcgtcagatgtgtataagagacaggggcggatgccattgagt (SEQ gtctcgtgggctcggagatgtgtataagagacagagcaaaaccgcaacccact ID NO: 527) (SEQ ID NO: 641) rs1698232 tcgtcggcagcgtcagatgtgtataagagacaggcacttctaagttattatgatagagt gtctcgtgggctcggagatgtgtataagagacagactcatatctcccaacacaaaact gatgtac (SEQ ID NO: 528) aaaa (SEQ ID NO: 642) rs670962 tcgtcggcagcgtcagatgtgtataagagacaggcagtaaatcaacccgctataaac gtctcgtgggctcggagatgtgtataagagacagtgaagcgtgaacttcctcagg g (SEQ ID NO: 529) (SEQ ID NO: 643) rs58445115 tcgtcggcagcgtcagatgtgtataagagacaggagcccttgccaatagtgaaa gtctcgtgggctcggagatgtgtataagagacagcacctgggaagagaggtgt (SEQ ID NO: 530) (SEQ ID NO: 644) rs59061318 tcgtcggcagcgtcagatgtgtataagagacagcagctagttctatatttacagacag gtctcgtgggctcggagatgtgtataagagacagtgaatatgtcttcagtgcttagcct agac (SEQ ID NO: 531) (SEQ ID NO: 645) rs6506015 tcgtcggcagcgtcagatgtgtataagagacagaatcctgtatctagtgccaatctag gtctcgtgggctcggagatgtgtataagagacagcctaaaaatcgttacttctcctttat aa (SEQ ID NO: 532) tttttttc (SEQ ID NO: 646) rs72634353 tcgtcggcagcgtcagatgtgtataagagacagagtatctataatagtgcgtggcaca gtctcgtgggctcggagatgtgtataagagacaggcctataacaatgtactagaacc ta (SEQ ID NO: 533) aagtattt (SEQ ID NO: 647) rs55677929 tcgtcggcagcgtcagatgtgtataagagacagggaagacccggcggga (SEQ gtctcgtgggctcggagatgtgtataagagacaggggtgtagggcaggggt ID NO: 534) (SEQ ID NO: 648) rs6135141 tcgtcggcagcgtcagatgtgtataagagacagtcctctcctgcttaatgtagtcac gtctcgtgggctcggagatgtgtataagagacagttcacaagcagagttgaaagact (SEQ ID NO: 535) (SEQ ID NO: 649) rs2050980 tcgtcggcagcgtcagatgtgtataagagacagatccaccagagaatacacaaatta gtctcgtgggctcggagatgtgtataagagacagaaaagactgtcagtgatatcttag tatgtatatat (SEQ ID NO: 536) gtaga (SEQ ID NO: 650) rs4815580 tcgtcggcagcgtcagatgtgtataagagacagggtacatgaccataataaatcagc gtctcgtgggctcggagatgtgtataagagacagggccaacattttgttttataatctg agg (SEQ ID NO: 537) cg (SEQ ID NO: 651) rs463397 tcgtcggcagcgtcagatgtgtataagagacagctctctctactgaattttgattttccatttc gtctcgtgggctcggagatgtgtataagagacagctaggatcaaagaagaatagaa (SEQ ID NO: 538) aaagtggt (SEQ ID NO: 652) rs7279689 tcgtcggcagcgtcagatgtgtataagagacagacactgagtattcccaatgtaaag gtctcgtgggctcggagatgtgtataagagacagacaataattgtacttatttatggag aaataat (SEQ ID NO: 539) tacatagtgat (SEQ ID NO: 653) rs5748211 tcgtcggcagcgtcagatgtgtataagagacagcctgggcatcgccct (SEQ gtctcgtgggctcggagatgtgtataagagacagcctaggaggtgacctcactaaa ID NO: 540) at (SEQ ID NO: 654) rs79114187 tcgtcggcagcgtcagatgtgtataagagacagggcccactgcactcacct (SEQ gtctcgtgggctcggagatgtgtataagagacagactatctacatcagtgcgagaga ID NO: 541) aag (SEQ ID NO: 655) rs13164 tcgtcggcagcgtcagatgtgtataagagacagcaggtagggaaagatttcttaagt gtctcgtgggctcggagatgtgtataagagacagtgtgcagagtcccccagg gag (SEQ ID NO: 542) (SEQ ID NO: 656) rs4633 tcgtcggcagcgtcagatgtgtataagagacagcctgcagcccatccacaac gtctcgtgggctcggagatgtgtataagagacagggcctccagcacgctc (SEQ (SEQ ID NO: 543) ID NO: 657) rs13303106 tcgtcggcagcgtcagatgtgtataagagacagactgtgagaggctcagaagga gtctcgtgggctcggagatgtgtataagagacaggggtagattccaggggctct (SEQ ID NO: 544) (SEQ ID NO: 658) rs35273536 tcgtcggcagcgtcagatgtgtataagagacagggggtcagcaggtggca (SEQ gtctcgtgggctcggagatgtgtataagagacagggaacactatctgaaatagaccc ID NO: 545) tcg (SEQ ID NO: 659) rs77129670 tcgtcggcagcgtcagatgtgtataagagacaggggagatgaaataagtaccaaaa gtctcgtgggctcggagatgtgtataagagacagcctgcaggtctttccgattctg tgagt (SEQ ID NO: 546) (SEQ ID NO: 660) rs17133064 tcgtcggcagcgtcagatgtgtataagagacaggcattgccactttggctttcg gtctcgtgggctcggagatgtgtataagagacaggattttaaaaccagagataattct (SEQ ID NO: 547) gaaaggaa (SEQ ID NO: 661) rs1161901 tcgtcggcagcgtcagatgtgtataagagacagcctctctaaaacttgatgattttaac gtctcgtgggctcggagatgtgtataagagacaggctggtcctcactgacatcc atgtaat (SEQ ID NO: 548) (SEQ ID NO: 662) rs77474447 tcgtcggcagcgtcagatgtgtataagagacaggacccacagccgtggt (SEQ gtctcgtgggctcggagatgtgtataagagacagtctaaccccgtcatgctgc ID NO: 549) (SEQ ID NO: 663) rs17756915 tcgtcggcagcgtcagatgtgtataagagacagaaatatatagagccgcacaccaa gtctcgtgggctcggagatgtgtataagagacaggtgcaatgttaactttattaattagt aaata (SEQ ID NO: 550) tgacttc (SEQ ID NO: 664) rs341697 tcgtcggcagcgtcagatgtgtataagagacagaaaaatggggcagaatgttgtca gtctcgtgggctcggagatgtgtataagagacagttgctgttccacaaaacatagctt (SEQ ID NO: 551) (SEQ ID NO: 665) rs10976019 tcgtcggcagcgtcagatgtgtataagagacaggagcaagttcggtctggct gtctcgtgggctcggagatgtgtataagagacaggctgattaattaggtgtttgttagc (SEQ ID NO: 552) ag (SEQ ID NO: 666) rs76408959 tcgtcggcagcgtcagatgtgtataagagacagcccattgattaaacaaatattcact gtctcgtgggctcggagatgtgtataagagacagcctcaccctccatccctca gagtac (SEQ ID NO: 553) (SEQ ID NO: 667) rs9734804 tcgtcggcagcgtcagatgtgtataagagacagcttcgggcctctggacc (SEQ gtctcgtgggctcggagatgtgtataagagacagcccaaggcgggcacct (SEQ ID NO: 554) ID NO: 668) rs12792188 tcgtcggcagcgtcagatgtgtataagagacagatctcccgtctcatcctgaaac gtctcgtgggctcggagatgtgtataagagacaggggtgctgcgcccaga (SEQ (SEQ ID NO: 555) ID NO: 669) rs11611246 tcgtcggcagcgtcagatgtgtataagagacaggtggaaggcatactgagtgaact gtctcgtgggctcggagatgtgtataagagacagggataaactagtggttctttgatct (SEQ ID NO: 556) ttatcttt (SEQ ID NO: 670) rs79782920 tcgtcggcagcgtcagatgtgtataagagacaggtgtctccattacattgcttgttttaattt gtctcgtgggctcggagatgtgtataagagacagaacatattgaacttatttttaaaag (SEQ ID NO: 557) ggggag (SEQ ID NO: 671) rs7989876 tcgtcggcagcgtcagatgtgtataagagacagtctgacatttcacagctggca gtctcgtgggctcggagatgtgtataagagacaggggggatctgccatacagc (SEQ ID NO: 558) (SEQ ID NO: 672) rs7982082 tcgtcggcagcgtcagatgtgtataagagacaggctcccaccagctactgtga gtctcgtgggctcggagatgtgtataagagacagtcaagccttgacttttaaagcaca (SEQ ID NO: 559) (SEQ ID NO: 673) rs77905703 tcgtcggcagcgtcagatgtgtataagagacaggttcacccagaagtcattccgta gtctcgtgggctcggagatgtgtataagagacagttgttgttgtcagcttagaatagtat (SEQ ID NO: 560) atcatat (SEQ ID NO: 674) rs59329234 tcgtcggcagcgtcagatgtgtataagagacaggaggcacagtgctttgtatttgat gtctcgtgggctcggagatgtgtataagagacagcccttttataatcttgttacttttatgt (SEQ ID NO: 561) tgaact (SEQ ID NO: 675) rs150926 tcgtcggcagcgtcagatgtgtataagagacagttagccactacctttttggctacta gtctcgtgggctcggagatgtgtataagagacagggggagagaaaaccgtatgtga (SEQ ID NO: 562) t (SEQ ID NO: 676) rs12450330 tcgtcggcagcgtcagatgtgtataagagacagcgttttacagcaagcgacagaa gtctcgtgggctcggagatgtgtataagagacagttttagatctctttcagttttggtttgc (SEQ ID NO: 563) (SEQ ID NO: 677) rs16948415 tcgtcggcagcgtcagatgtgtataagagacagatgtgatgtgctctaggaaaatgc gtctcgtgggctcggagatgtgtataagagacagggagttataaaaaagaacagaa (SEQ ID NO: 564) ggtcatgat (SEQ ID NO: 678) rs11878153 tcgtcggcagcgtcagatgtgtataagagacaggccttgtgagaacagactaataca gtctcgtgggctcggagatgtgtataagagacaggcctccttcactcgcagt (SEQ gata (SEQ ID NO: 565) ID NO: 679) rs2279796 tcgtcggcagcgtcagatgtgtataagagacagggtaggtgtggtcaggtcga gtctcgtgggctcggagatgtgtataagagacaggcacaagcggtcaacagc (SEQ ID NO: 566) (SEQ ID NO: 680) rs6074167 tcgtcggcagcgtcagatgtgtataagagacagacctccctcttgggatgca gtctcgtgggctcggagatgtgtataagagacaggggtttatcaaaaggtttgctgc (SEQ ID NO: 567) (SEQ ID NO: 681) rs2823170 tcgtcggcagcgtcagatgtgtataagagacagggagaataatagttaattaatccac gtctcgtgggctcggagatgtgtataagagacagcgctaaaaaaggaagaactagg gaagca (SEQ ID NO: 568) aaagat (SEQ ID NO: 682) rs9984697 tcgtcggcagcgtcagatgtgtataagagacagagagtctcaattattgctcagttag gtctcgtgggctcggagatgtgtataagagacagaatatctatccagagtatcgatta gat (SEQ ID NO: 569) atcttcataaaa (SEQ ID NO: 683) rs17809319 tcgtcggcagcgtcagatgtgtataagagacagggtgacctgcgttacttgcttat gtctcgtgggctcggagatgtgtataagagacagacctagtacgtcatttataatgaa (SEQ ID NO: 570) aattgc (SEQ ID NO: 684)

[0097] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

REFERENCES

[0098] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. [0099] U.S. Pat. No. 4,683,195 [0100] U.S. Pat. No. 4,683,202 [0101] U.S. Pat. No. 4,800,159 [0102] U.S. Pat. No. 6,664,079 [0103] U.S. Pat. No. 8,612,161 [0104] U.S. Pat. No. 8,623,598 [0105] U.S. Pat. No. 9,284,602 [0106] U.S. Pat. Publn. No. 2009/0026082 [0107] U.S. Pat. Publn. No. 2010/0137143 [0108] U.S. Pat. Publn. No. 2010/0282617 [0109] U.S. Pat. Publn. No. 2016/0326600 [0110] U.S. Pat. Publn. No. 2016/0340727 [0111] U.S. Pat. Publn. No. 2017/0029875 [0112] Margulies et al., Nature, 437:376-380, 2005. [0113] McPherson et al., editors, PCR: A Practical Approach (IRL Press, Oxford, 1991). [0114] McPherson et al., editors, PCR2: A Practical Approach (IRL Press, Oxford, 1995). [0115] Oyola et al., BMC Genomics, 13:1, 2012. [0116] Pareek et al., Sequencing technologies and genome sequencing, J. Appl. Genet., 52(4):413-435, 2011. [0117] Thudi et al., Current state-of-art of sequencing technologies for plant genomics research, Brief Funct. Genomics, 11(1):3-11, 2012. [0118] Wang et al., Modular probes for enriching and detecting complex nucleic acid sequences, Nature Chemistry, DOI: 10.1038/NCHEM.2820, Published online Jul. 17, 2017. [0119] Wang and Zhang, Simulation-guided DNA probe design for consistently ultraspecific hybridization, Nature Chemistry, 7:545-53, 2015. [0120] Wu et al., Continuously tunable nucleic acid hybridization probes, Nature Methods, 12:1191-96, 2015. [0121] Zhang et al., Optimizing the specificity of nucleic acid hybridization, Nature Chemistry, 4:208-14, 2012.

Sequence CWU 1

1

687185DNAArtificial SequenceSynthetic oligonucleotide 1aaataatcag gagaaggaga tggcatgttt gttggtgatt ccaaggagct ctctcagtca 60tgatcctgta catttgctct gcctt 85287DNAArtificial SequenceSynthetic oligonucleotide 2ggagcagcgt ctctgccatc gtcctcgtcc atgtcctggt cacacttcca gattaactac 60ttcgaacctg tacatttgct ctgcctt 87381DNAArtificial SequenceSynthetic oligonucleotide 3aggtaaatat ttaccacctc ttggtgttta ttttaccgtc tatatacaag agcaccgcaa 60cctgtacatt tgctctgcct t 81483DNAArtificial SequenceSynthetic oligonucleotide 4ctttcagtca gatgtatatg catttgggat tgttctgtat gaattgatga gtatgtgcgg 60ttcctgtaca tttgctctgc ctt 83585DNAArtificial SequenceSynthetic oligonucleotide 5tatctgctaa gaaacagaca tccatataca gagatgaaaa tgatgatttt tgatacatca 60actgcctgta catttgctct gcctt 85685DNAArtificial SequenceSynthetic oligonucleotide 6atcttgcctt gccttccacc ctaataccag cataatctac taaagcttct ctacaactga 60tacgcctgta catttgctct gcctt 85784DNAArtificial SequenceSynthetic oligonucleotide 7tgtccagtga tatggttata atgtgaaaca aaactcacct gggtcacttt tactgcagac 60cttcctgtac atttgctctg cctt 84883DNAArtificial SequenceSynthetic oligonucleotide 8attaaaggcg ccgccgggcg gctcccgctg ccatcctggc tctcctgcgc actgagagat 60aacctgtaca tttgctctgc ctt 83987DNAArtificial SequenceSynthetic oligonucleotide 9gatttgcgtt ctgcactatg acataatttg gccttcaggg tgagttgttt tccgatatat 60atgtctcctg tacatttgct ctgcctt 871084DNAArtificial SequenceSynthetic oligonucleotide 10agacttcacc ttgtgatctg cagggactga ccttagtgtt gttgtgttgg caatagtctt 60ggacctgtac atttgctctg cctt 841188DNAArtificial SequenceSynthetic oligonucleotide 11agagatctcc aaagacactc cacggaatga gggcttgttg cccttgtctt tatgttaata 60tcaacagcct gtacatttgc tctgcctt 881283DNAArtificial SequenceSynthetic oligonucleotide 12cctatacaat tgagatgttg ggggaaccac aacataactc attcagactg tgacgagctt 60gacctgtaca tttgctctgc ctt 831383DNAArtificial SequenceSynthetic oligonucleotide 13tttttattgt atatgcatgc acatcccaag gaccaagaga ccagctacac cgcatgtgac 60gacctgtaca tttgctctgc ctt 831484DNAArtificial SequenceSynthetic oligonucleotide 14gcaggcatca aagtgcagga cgtccggctg aatggctccg cagctggcca aagctgccat 60tcccctgtac atttgctctg cctt 841585DNAArtificial SequenceSynthetic oligonucleotide 15tctgcattcc ctgtcactgc gtcactggcc ttcagacaga gccaaggtgc ttgattgcta 60caatcctgta catttgctct gcctt 851683DNAArtificial SequenceSynthetic oligonucleotide 16gcccaagtgt ttctctggca tctgttggtg tctggatcca ccactctact ggtgcttcgt 60cacctgtaca tttgctctgc ctt 831787DNAArtificial SequenceSynthetic oligonucleotide 17gttcagattt cactgcctca tgttgatatt tctttccaga tgacgactgt ttaatctctc 60acacctcctg tacatttgct ctgcctt 871886DNAArtificial SequenceSynthetic oligonucleotide 18tagacacatt gtcatcatgg acaggcggtg gatacgtgcc gtctagctga tacctaacta 60tagaacctgt acatttgctc tgcctt 861985DNAArtificial SequenceSynthetic oligonucleotide 19gcttgtcttt gaaacttctt ggcaaatcgg ttaagatctg ggaatcgacg gttcatacct 60caatcctgta catttgctct gcctt 852082DNAArtificial SequenceSynthetic oligonucleotide 20tacctcccat attggggcct acagaacaaa ttatatcaga aagcaagatt gataccgttg 60acctgtacat ttgctctgcc tt 822182DNAArtificial SequenceSynthetic oligonucleotide 21tgccaatgac cacagtgtcg ggccccgcat ccagtgacga gggcgtggtg ggagaaccag 60acctgtacat ttgctctgcc tt 822283DNAArtificial SequenceSynthetic oligonucleotide 22atccccctta aaatcacgct cacttgccgc gcataggcca tctcgatgtt gacgacgaac 60agcctgtaca tttgctctgc ctt 832386DNAArtificial SequenceSynthetic oligonucleotide 23ggggaggtga agctgtctat ctcctacaaa aacaataaac tcttcatcat attaacgtca 60ataaacctgt acatttgctc tgcctt 862487DNAArtificial SequenceSynthetic oligonucleotide 24cgagattttt ttccttccaa tatattctac ataagttccc ggaaagtccc agaatggatc 60taaaagcctg tacatttgct ctgcctt 872582DNAArtificial SequenceSynthetic oligonucleotide 25cttttcctta aaaagaaaaa gaaagggagt cattaagcaa ccacgtatca ccgatcattg 60tcctgtacat ttgctctgcc tt 822685DNAArtificial SequenceSynthetic oligonucleotide 26tgtgtgttat gatttctgtt gcagagttgt gaaaaccgtg gctgtgaaaa caccagaggt 60cttccctgta catttgctct gcctt 852784DNAArtificial SequenceSynthetic oligonucleotide 27ggccctgcaa atgccctcat cagaagcccc gttgccctcc tggagtgagc actcgaagat 60cgacctgtac atttgctctg cctt 842884DNAArtificial SequenceSynthetic oligonucleotide 28gctactccag gcacacgccg cacatcctgc aggcagagag taagaggaag tcgatacagc 60ctacctgtac atttgctctg cctt 842983DNAArtificial SequenceSynthetic oligonucleotide 29gatctctcct gagtcctcac taacaacagg gggtagattt attgttttca agccaagtat 60cacctgtaca tttgctctgc ctt 833081DNAArtificial SequenceSynthetic oligonucleotide 30ccactagccc tggttcaggt cagggatgcc atgtcgtcgg ggcccaggca aatcttttcg 60cctgtacatt tgctctgcct t 813184DNAArtificial SequenceSynthetic oligonucleotide 31tggcagcctc actgtgcgga gcatggagcc acacatggtg taggcacagt taagttattt 60atacctgtac atttgctctg cctt 843283DNAArtificial SequenceSynthetic oligonucleotide 32aagtacccca aagtgtgagg gccttccctc tgccgcacat catcgagaac ggtccggtcg 60ctcctgtaca tttgctctgc ctt 833385DNAArtificial SequenceSynthetic oligonucleotide 33gtgcttctga aactgttatc ttcccaggag caatctgggg atagaaagca tattagtgtg 60gagacctgta catttgctct gcctt 853486DNAArtificial SequenceSynthetic oligonucleotide 34gggccagtct ttaaatgctt cctggaaaat gttactacct atgaaataaa cttctggcag 60tggaccctgt acatttgctc tgcctt 863584DNAArtificial SequenceSynthetic oligonucleotide 35aaggaagcag cgtgcagtgc cattccttcc tccacgtaag cctcgttagg atccagtcaa 60cttcctgtac atttgctctg cctt 843684DNAArtificial SequenceSynthetic oligonucleotide 36attgggggta tactggaaaa gtatttttgg tgttgaagtt tgggctgtga gcgagccttg 60tcacctgtac atttgctctg cctt 843782DNAArtificial SequenceSynthetic oligonucleotide 37gggatgtttc ttgtcctcgc tcaagacaga attcgagagt gagccagtgg cggtggaagg 60acctgtacat ttgctctgcc tt 823884DNAArtificial SequenceSynthetic oligonucleotide 38tttgacacca ataaaacgga gtgccactga agggttttga gctgaggaga gtccagtgaa 60ctacctgtac atttgctctg cctt 843984DNAArtificial SequenceSynthetic oligonucleotide 39accgtacctc tccccgacgt gggcaggcgt gagttgtcag tttagagaac aacataatcg 60tagcctgtac atttgctctg cctt 844083DNAArtificial SequenceSynthetic oligonucleotide 40ctgcttctag ggttgggatc tcccagggaa gaccgggctt gccatgtgca acgaggcctt 60ctcctgtaca tttgctctgc ctt 834188DNAArtificial SequenceSynthetic oligonucleotide 41gtcattttgc tgtttgtttt ctatatgcag tataacattt ttgtctctta atatgtaatc 60agctgatcct gtacatttgc tctgcctt 884283DNAArtificial SequenceSynthetic oligonucleotide 42taggagacag agaatgttct gtgggaccac aaccgagaca gaagagctct aatgccggcc 60gccctgtaca tttgctctgc ctt 834385DNAArtificial SequenceSynthetic oligonucleotide 43cctgtaacac acgcccacag gggcttcagg aactataaac attcaccctt ttctcttgga 60tatgcctgta catttgctct gcctt 854483DNAArtificial SequenceSynthetic oligonucleotide 44tatttgataa attaacccta gaacaactat ctgcactcag aaacacatac ggaacgcatt 60agcctgtaca tttgctctgc ctt 834585DNAArtificial SequenceSynthetic oligonucleotide 45gagcatcctg aagcaattct gtttgtaatc ctggaagtag taactgggaa cctatactca 60ccagcctgta catttgctct gcctt 854685DNAArtificial SequenceSynthetic oligonucleotide 46tgaattattt ttcttcccct tcatttttgt ttaagctcta ttgttttttg taccattcga 60gccacctgta catttgctct gcctt 854786DNAArtificial SequenceSynthetic oligonucleotide 47agaacaatgt ccacatgttt cctctgtgcc attactaaga tggtcccctt accattggcg 60gaagccctgt acatttgctc tgcctt 864884DNAArtificial SequenceSynthetic oligonucleotide 48catcccaccc tgtctcactg gagccaggat ccatgaggtc ccgtgagcta ggcttcagaa 60ggccctgtac atttgctctg cctt 844982DNAArtificial SequenceSynthetic oligonucleotide 49tccatcctaa aggacttaca gtttcttaga ataacatgga gtgatttttc ggagtgactg 60tcctgtacat ttgctctgcc tt 825085DNAArtificial SequenceSynthetic oligonucleotide 50gaaaacagtc aaaatggctg tcgacaatga aatggataca tcagttgaga ctcaggcaat 60gtgacctgta catttgctct gcctt 855184DNAArtificial SequenceSynthetic oligonucleotide 51gaaagactaa taattttgcc catgatcacc tcaccatctc actctgaaat ggcggctctc 60aggcctgtac atttgctctg cctt 845284DNAArtificial SequenceSynthetic oligonucleotide 52agtagtcggc atggtgctga gcaccctccg ggaaccgtcc ctacctgaaa ggctaccgag 60ccgcctgtac atttgctctg cctt 845386DNAArtificial SequenceSynthetic oligonucleotide 53aattctagct ccaaaatctg ggctcctgac cacaatgtta gaggtgcctt gcattagtgt 60ggagacctgt acatttgctc tgcctt 865484DNAArtificial SequenceSynthetic oligonucleotide 54gaaccgtcac caggtccttt attgcctctt ccaacaatag aaattatcca tcgcaagatc 60gtgcctgtac atttgctctg cctt 845586DNAArtificial SequenceSynthetic oligonucleotide 55agtcctaacc taggttacag cccatcacag ctggagcagg tgatgcttag aacctgatac 60accatcctgt acatttgctc tgcctt 865682DNAArtificial SequenceSynthetic oligonucleotide 56gtcaggctta agaggcaggg ccacctaaac gtctactgag aacacagggt attggcgagc 60ccctgtacat ttgctctgcc tt 825784DNAArtificial SequenceSynthetic oligonucleotide 57ttcagatatg actagggaat gtttagaaag tacacgccac atgcttcatg actaggcgag 60gtccctgtac atttgctctg cctt 845885DNAArtificial SequenceSynthetic oligonucleotide 58ctccaggtat agatgcaagt aggctggtag atttaatgag gagacaaact cgttcagttc 60agcccctgta catttgctct gcctt 855985DNAArtificial SequenceSynthetic oligonucleotide 59ctgggtgtaa agtttctgtg caaacctttg ctacagtgcg tgccgagtca aagacatccg 60cgagcctgta catttgctct gcctt 856082DNAArtificial SequenceSynthetic oligonucleotide 60gacctgtagt cacaagtgta gagagtttga gcttcgactt agaaaggcac accacgatac 60tcctgtacat ttgctctgcc tt 826183DNAArtificial SequenceSynthetic oligonucleotide 61ttcactggcg atcaacagta accaataaaa ttcactcatg aatcaacagt ccttccggta 60ggcctgtaca tttgctctgc ctt 836284DNAArtificial SequenceSynthetic oligonucleotide 62tctctgtagt caatttgatt tttatcaagt tgcactaaat attttaagac cctatcacta 60gagcctgtac atttgctctg cctt 846387DNAArtificial SequenceSynthetic oligonucleotide 63cagcctgtgt tcaggatctc acaaagtctc tcatgaaaat agttgtgggc acctatacga 60tgaatccctg tacatttgct ctgcctt 876483DNAArtificial SequenceSynthetic oligonucleotide 64ttgttctcat ctctcagaag cccttctgtg gcccaaacat tattgaccat cgccagactt 60gccctgtaca tttgctctgc ctt 836583DNAArtificial SequenceSynthetic oligonucleotide 65gaagcctagg tatgtaaatt acaggcttgc agaagtaaat gcaggtggct cgccatgatg 60tccctgtaca tttgctctgc ctt 836685DNAArtificial SequenceSynthetic oligonucleotide 66ggacgagccc cagaaaagtg gaagaagact aatgatgcca ccttggcccc gatgatagtt 60ccagcctgta catttgctct gcctt 856782DNAArtificial SequenceSynthetic oligonucleotide 67tgccctcgtc cctactggta agaggcataa ggtggggaag ggcctaagtg ctcaccagtc 60tcctgtacat ttgctctgcc tt 826889DNAArtificial SequenceSynthetic oligonucleotide 68cttaaaacta aaacaggaaa aaaaaatcaa aaccgtaaca aatcagtaat cacgatgtta 60cgtggctgcc tgtacatttg ctctgcctt 896984DNAArtificial SequenceSynthetic oligonucleotide 69aaatcagtaa aatgtttaca agcaatatct tttacgatct taaaactaag acgaccatcc 60tcacctgtac atttgctctg cctt 847085DNAArtificial SequenceSynthetic oligonucleotide 70ctacataaca gaattcagta tgcagtcatg atacatactc tcagcaaagt tatcgagcca 60ttggcctgta catttgctct gcctt 857182DNAArtificial SequenceSynthetic oligonucleotide 71tcctctcagt ctctgagctc tgtagaggag cctcaggggc agatgcaacc gaagaaggta 60tcctgtacat ttgctctgcc tt 827285DNAArtificial SequenceSynthetic oligonucleotide 72acacaaaact aaaagcactt ttaatatttc ttcaaaactt ctttcaattc caacgagaga 60gcgacctgta catttgctct gcctt 857383DNAArtificial SequenceSynthetic oligonucleotide 73agccactcca ctcctaggta tctgcccgag agacatgaaa gcacaaggac catcggtcgt 60cgcctgtaca tttgctctgc ctt 837481DNAArtificial SequenceSynthetic oligonucleotide 74tgatccccaa cagagagagg tacccgggat cttctgacgt ggttcatgtg cgcatcgttt 60cctgtacatt tgctctgcct t 817585DNAArtificial SequenceSynthetic oligonucleotide 75tccgaattct ccaactttcc tcccagcacg ggtctgccct tgggactcgg gcttcagtca 60tgttcctgta catttgctct gcctt 857684DNAArtificial SequenceSynthetic oligonucleotide 76tttcctttct ttcttccaaa ctcctcttaa tattggtatt ttgacctcct agaacgccag 60ggacctgtac atttgctctg cctt 847785DNAArtificial SequenceSynthetic oligonucleotide 77acatagaagg tgttcagtaa atatttcctg acagtaggag ttgatgaatg caggtatgga 60gcagcctgta catttgctct gcctt 857884DNAArtificial SequenceSynthetic oligonucleotide 78tcatggccgg tggccggttc tcaccccttt tgctcctaac agacacgcag agcctaacac 60tgccctgtac atttgctctg cctt 847985DNAArtificial SequenceSynthetic oligonucleotide 79ggagatactg acaattgcaa gttgggctga tatgcatgaa aacagaaaat atcgagaacc 60cgcacctgta catttgctct gcctt 858085DNAArtificial SequenceSynthetic oligonucleotide 80taacaaagac tagcttatac tacccacact ttcctgtcat ttttcttttc ggtagttcca 60attgcctgta catttgctct gcctt 858183DNAArtificial SequenceSynthetic oligonucleotide 81aacattttgt tttataatct gcgtctgata ataccgatat acaaactctg caatggagtt 60ggcctgtaca tttgctctgc ctt 838285DNAArtificial SequenceSynthetic oligonucleotide 82agatggtgaa gtaaagatga ataacatgaa gcacatttga atgctattgg ccttgattct 60cgttcctgta catttgctct gcctt 858388DNAArtificial SequenceSynthetic oligonucleotide 83tagtgatatt tcaatacata taatgtatag tgatcagtgt aattagcata atgtacgatt 60attagaccct gtacatttgc tctgcctt 888487DNAArtificial SequenceSynthetic oligonucleotide 84ctttctctag gtgccgtaca tgttagtggg agctccttat ttcctggatt ggagaggtag 60gtcttgcctg

tacatttgct ctgcctt 878586DNAArtificial SequenceSynthetic oligonucleotide 85aactctcagt ttgggccact gctctccagt tgcctggagt tttaagtctt acgacctgtg 60ataggcctgt acatttgctc tgcctt 868683DNAArtificial SequenceSynthetic oligonucleotide 86atggccaagc cttggctgtt gagtaggcac tgcccagttg tgctgtatgg ttgagccatg 60gtcctgtaca tttgctctgc ctt 838785DNAArtificial SequenceSynthetic oligonucleotide 87tcccgggctc cgcatgctgc agcacatggt tcaggatgcg ctgctccttg attccttact 60cctacctgta catttgctct gcctt 858878DNAArtificial SequenceSynthetic oligonucleotide 88gaaggacccc agctccacca accaacaaag gcacagtccg tcccacccac cttattgcct 60gtacatttgc tctgcctt 788987DNAArtificial SequenceSynthetic oligonucleotide 89gaaatagacc ctcgacagac ccaaaggggc ccacgtgatg cggtggtgac gttagagaag 60taaggacctg tacatttgct ctgcctt 879083DNAArtificial SequenceSynthetic oligonucleotide 90cccagatttt gctattccat acagttgact ggacatgaac tcattttggt ggcgtacgga 60ggcctgtaca tttgctctgc ctt 839183DNAArtificial SequenceSynthetic oligonucleotide 91attctgaaag gaatgaaaat ggggtttaaa tgtccttaag gtccagaggg catcttccag 60ttcctgtaca tttgctctgc ctt 839288DNAArtificial SequenceSynthetic oligonucleotide 92attctgaaga tttatcgtga aaaaaaaaga atgtacaatc ttttattaat atcgaatcca 60acatactcct gtacatttgc tctgcctt 889382DNAArtificial SequenceSynthetic oligonucleotide 93caacctgccc ctccctgacc cggggccccc tttcctccag ggcccagtct cgcctgctga 60acctgtacat ttgctctgcc tt 829485DNAArtificial SequenceSynthetic oligonucleotide 94gttgacttct tttaaaatat gatcttcaca attaccatcc aatcaaattg tgagaagctg 60cgttcctgta catttgctct gcctt 859586DNAArtificial SequenceSynthetic oligonucleotide 95atagctttac cattttacct tgctcaatac gcaccccaga caaagagaca cgtgatatct 60cccaacctgt acatttgctc tgcctt 869682DNAArtificial SequenceSynthetic oligonucleotide 96tttgttagca gggttggatc taaccagtga tgtgcggcat gtcagtgtga gcttacattt 60ccctgtacat ttgctctgcc tt 829789DNAArtificial SequenceSynthetic oligonucleotide 97cctcgttacc tgcttctcat ctgtgatgct ccccagatct ctgcaacatt tgccgaggta 60taagcggacc tgtacatttg ctctgcctt 899884DNAArtificial SequenceSynthetic oligonucleotide 98gcctggggcc gggcggcagg ggcgcgcagg gtggcggccc agaggcctct attagtagta 60gaccctgtac atttgctctg cctt 849979DNAArtificial SequenceSynthetic oligonucleotide 99gagagagggt gctaggctgc tggcccagca aggcctcaag ggaagcaggg acattagtcc 60tgtacatttg ctctgcctt 7910086DNAArtificial SequenceSynthetic oligonucleotide 100ggggttgggg gggtggtgtt gaggtatgtg taagtctatt gctcatgata gccaccaatt 60atcgacctgt acatttgctc tgcctt 8610187DNAArtificial SequenceSynthetic oligonucleotide 101aggcgggaac ataaactaac aaaaaagtat gtcacagcac agtgacatgt ttctggcagt 60acatctcctg tacatttgct ctgcctt 8710280DNAArtificial SequenceSynthetic oligonucleotide 102tgatgggagc acacccccca atgaccctgc ccccacacct gcaaaagccc ttatagactc 60ctgtacattt gctctgcctt 8010385DNAArtificial SequenceSynthetic oligonucleotide 103ttaaagcaca ttaaagctca ttagccacta tgtcgaggcc ttatctaatc aagtccatcc 60tgaccctgta catttgctct gcctt 8510485DNAArtificial SequenceSynthetic oligonucleotide 104tagtatatca tataaaaata aagacatcac ccaacataaa aacatcaccc ttcgaggtgt 60tggccctgta catttgctct gcctt 8510584DNAArtificial SequenceSynthetic oligonucleotide 105atgttgaact cttttgtcaa aagccccttg ttggaaaagt ctagagactt cgcatgacat 60cttcctgtac atttgctctg cctt 8410687DNAArtificial SequenceSynthetic oligonucleotide 106aaaccgtatg tgatctagca atggaggaga gggtcagaag tccccagtac cttgcataga 60gaaaatcctg tacatttgct ctgcctt 8710786DNAArtificial SequenceSynthetic oligonucleotide 107tcaaatttcc cgtgatcatt actgcccatt tcccaaaata tctcattgca catcttaagc 60cacggcctgt acatttgctc tgcctt 8610884DNAArtificial SequenceSynthetic oligonucleotide 108ggtcatgata agtaagcagt gaaacaaagt agacatatga atcagtaaaa cgttacctct 60agacctgtac atttgctctg cctt 8410987DNAArtificial SequenceSynthetic oligonucleotide 109cttcactcgc agtaaatgtc tatttctcct gtttcattaa aaaggttgag ttaactacac 60acaaggcctg tacatttgct ctgcctt 8711083DNAArtificial SequenceSynthetic oligonucleotide 110ctctgcccac ggtatacctg ggagagtgca ggtccttcag aaaggtgagt tcggtcatcc 60ttcctgtaca tttgctctgc ctt 8311188DNAArtificial SequenceSynthetic oligonucleotide 111tgatcatatg gtttttgttt ttaattctgt ttatatggtg aatcacactt acagtccgat 60aacaccgcct gtacatttgc tctgcctt 8811289DNAArtificial SequenceSynthetic oligonucleotide 112agatagatga cttagaggcc cttgggtgta acagagagtc agtcttccca aatagaatca 60tctcatgacc tgtacatttg ctctgcctt 8911394DNAArtificial SequenceSynthetic oligonucleotide 113aatcttcata aaacctcagt gaatactctt ttttcctgtt aaaaaaatct taataataac 60gaattataac cggcctgtac atttgctctg cctt 9411485DNAArtificial SequenceSynthetic oligonucleotide 114cttgcttatg aacactaatt tcatatataa aacaaaaaat ttattgtgat aaccactgca 60tatgcctgta catttgctct gcctt 8511546DNAArtificial SequenceSynthetic oligonucleotide 115tgactgagag agctccttgg aatcaccaac aaacatgcca tctcct 4611653DNAArtificial SequenceSynthetic oligonucleotide 116aagtagttaa tctggaagtg tgaccaggac atggacgagg acgatggcag aga 5311742DNAArtificial SequenceSynthetic oligonucleotide 117cggtgctctt gtatatagac ggtaaaataa acaccaagag gt 4211846DNAArtificial SequenceSynthetic oligonucleotide 118gcacatactc atcaattcat acagaacaat cccaaatgca tataca 4611947DNAArtificial SequenceSynthetic oligonucleotide 119tgatgtatca aaaatcatca ttttcatctc tgtatatgga tgtctgt 4712049DNAArtificial SequenceSynthetic oligonucleotide 120tcagttgtag agaagcttta gtagattatg ctggtattag ggtggaagg 4912147DNAArtificial SequenceSynthetic oligonucleotide 121tctgcagtaa aagtgaccca ggtgagtttt gtttcacatt ataacca 4712246DNAArtificial SequenceSynthetic oligonucleotide 122tctcagtgcg caggagagcc aggatggcag cgggagccgc ccggcg 4612351DNAArtificial SequenceSynthetic oligonucleotide 123atatatatcg gaaaacaact caccctgaag gccaaattat gtcatagtgc a 5112448DNAArtificial SequenceSynthetic oligonucleotide 124agactattgc caacacaaca acactaaggt cagtccctgc agatcaca 4812551DNAArtificial SequenceSynthetic oligonucleotide 125tgatattaac ataaagacaa gggcaacaag ccctcattcc gtggagtgtc t 5112644DNAArtificial SequenceSynthetic oligonucleotide 126gctcgtcaca gtctgaatga gttatgttgt ggttccccca acat 4412743DNAArtificial SequenceSynthetic oligonucleotide 127cacatgcggt gtagctggtc tcttggtcct tgggatgtgc atg 4312849DNAArtificial SequenceSynthetic oligonucleotide 128tggcagcttt ggccagctgc ggagccattc agccggacgt cctgcactt 4912950DNAArtificial SequenceSynthetic oligonucleotide 129tagcaatcaa gcaccttggc tctgtctgaa ggccagtgac gcagtgacag 5013048DNAArtificial SequenceSynthetic oligonucleotide 130gaagcaccag tagagtggtg gatccagaca ccaacagatg ccagagaa 4813149DNAArtificial SequenceSynthetic oligonucleotide 131gtgagagatt aaacagtcgt catctggaaa gaaatatcaa catgaggca 4913249DNAArtificial SequenceSynthetic oligonucleotide 132atagttaggt atcagctaga cggcacgtat ccaccgcctg tccatgatg 4913349DNAArtificial SequenceSynthetic oligonucleotide 133aggtatgaac cgtcgattcc cagatcttaa ccgatttgcc aagaagttt 4913445DNAArtificial SequenceSynthetic oligonucleotide 134cggtatcaat cttgctttct gatataattt gttctgtagg cccca 4513547DNAArtificial SequenceSynthetic oligonucleotide 135gttctcccac cacgccctcg tcactggatg cggggcccga cactgtg 4713647DNAArtificial SequenceSynthetic oligonucleotide 136ttcgtcgtca acatcgagat ggcctatgcg cggcaagtga gcgtgat 4713752DNAArtificial SequenceSynthetic oligonucleotide 137ttgacgttaa tatgatgaag agtttattgt ttttgtagga gatagacagc tt 5213849DNAArtificial SequenceSynthetic oligonucleotide 138tagatccatt ctgggacttt ccgggaactt atgtagaata tattggaag 4913941DNAArtificial SequenceSynthetic oligonucleotide 139atcggtgata cgtggttgct taatgactcc ctttcttttt c 4114047DNAArtificial SequenceSynthetic oligonucleotide 140acctctggtg ttttcacagc cacggttttc acaactctgc aacagaa 4714151DNAArtificial SequenceSynthetic oligonucleotide 141tcttcgagtg ctcactccag gagggcaacg gggcttctga tgagggcatt t 5114249DNAArtificial SequenceSynthetic oligonucleotide 142ctgtatcgac ttcctcttac tctctgcctg caggatgtgc ggcgtgtgc 4914346DNAArtificial SequenceSynthetic oligonucleotide 143acttggcttg aaaacaataa atctaccccc tgttgttagt gaggac 4614446DNAArtificial SequenceSynthetic oligonucleotide 144aagatttgcc tgggccccga cgacatggca tccctgacct gaacca 4614550DNAArtificial SequenceSynthetic oligonucleotide 145aataacttaa ctgtgcctac accatgtgtg gctccatgct ccgcacagtg 5014646DNAArtificial SequenceSynthetic oligonucleotide 146accggaccgt tctcgatgat gtgcggcaga gggaaggccc tcacac 4614748DNAArtificial SequenceSynthetic oligonucleotide 147cacactaata tgctttctat ccccagattg ctcctgggaa gataacag 4814851DNAArtificial SequenceSynthetic oligonucleotide 148actgccagaa gtttatttca taggtagtaa cattttccag gaagcattta a 5114948DNAArtificial SequenceSynthetic oligonucleotide 149tgactggatc ctaacgaggc ttacgtggag gaaggaatgg cactgcac 4815047DNAArtificial SequenceSynthetic oligonucleotide 150aaggctcgct cacagcccaa acttcaacac caaaaatact tttccag 4715145DNAArtificial SequenceSynthetic oligonucleotide 151tccaccgcca ctggctcact ctcgaattct gtcttgagcg aggac 4515245DNAArtificial SequenceSynthetic oligonucleotide 152tcactggact ctcctcagct caaaaccctt cagtggcact ccgtt 4515348DNAArtificial SequenceSynthetic oligonucleotide 153gattatgttg ttctctaaac tgacaactca cgcctgccca cgtcgggg 4815447DNAArtificial SequenceSynthetic oligonucleotide 154ggcctcgttg cacatggcaa gcccggtctt ccctgggaga tcccaac 4715551DNAArtificial SequenceSynthetic oligonucleotide 155gctgattaca tattaagaga caaaaatgtt atactgcata tagaaaacaa a 5115646DNAArtificial SequenceSynthetic oligonucleotide 156ccggcattag agctcttctg tctcggttgt ggtcccacag aacatt 4615748DNAArtificial SequenceSynthetic oligonucleotide 157tccaagagaa aagggtgaat gtttatagtt cctgaagccc ctgtgggc 4815841DNAArtificial SequenceSynthetic oligonucleotide 158tgcgttccgt atgtgtttct gagtgcagat agttgttcta g 4115949DNAArtificial SequenceSynthetic oligonucleotide 159tgagtatagg ttcccagtta ctacttccag gattacaaac agaattgct 4916044DNAArtificial SequenceSynthetic oligonucleotide 160tcgaatggta caaaaaacaa tagagcttaa acaaaaatga aggg 4416149DNAArtificial SequenceSynthetic oligonucleotide 161ccgccaatgg taaggggacc atcttagtaa tggcacagag gaaacatgt 4916249DNAArtificial SequenceSynthetic oligonucleotide 162tctgaagcct agctcacggg acctcatgga tcctggctcc agtgagaca 4916345DNAArtificial SequenceSynthetic oligonucleotide 163tcactccgaa aaatcactcc atgttattct aagaaactgt aagtc 4516446DNAArtificial SequenceSynthetic oligonucleotide 164attgcctgag tctcaactga tgtatccatt tcattgtcga cagcca 4616542DNAArtificial SequenceSynthetic oligonucleotide 165agagccgcca tttcagagtg agatggtgag gtgatcatgg gc 4216649DNAArtificial SequenceSynthetic oligonucleotide 166tcggtagcct ttcaggtagg gacggttccc ggagggtgct cagcaccat 4916748DNAArtificial SequenceSynthetic oligonucleotide 167cacactaatg caaggcacct ctaacattgt ggtcaggagc ccagattt 4816849DNAArtificial SequenceSynthetic oligonucleotide 168atcttgcgat ggataatttc tattgttgga agaggcaata aaggacctg 4916948DNAArtificial SequenceSynthetic oligonucleotide 169tgtatcaggt tctaagcatc acctgctcca gctgtgatgg gctgtaac 4817045DNAArtificial SequenceSynthetic oligonucleotide 170cgccaatacc ctgtgttctc agtagacgtt taggtggccc tgcct 4517144DNAArtificial SequenceSynthetic oligonucleotide 171tcgcctagtc atgaagcatg tggcgtgtac tttctaaaca ttcc 4417247DNAArtificial SequenceSynthetic oligonucleotide 172gaactgaacg agtttgtctc ctcattaaat ctaccagcct acttgca 4717348DNAArtificial SequenceSynthetic oligonucleotide 173cggatgtctt tgactcggca cgcactgtag caaaggtttg cacagaaa 4817445DNAArtificial SequenceSynthetic oligonucleotide 174tcgtggtgtg cctttctaag tcgaagctca aactctctac acttg 4517548DNAArtificial SequenceSynthetic oligonucleotide 175ccggaaggac tgttgattca tgagtgaatt ttattggtta ctgttgat 4817646DNAArtificial SequenceSynthetic oligonucleotide 176agtgataggg tcttaaaata tttagtgcaa cttgataaaa atcaaa 4617751DNAArtificial SequenceSynthetic oligonucleotide 177catcgtatag gtgcccacaa ctattttcat gagagacttt gtgagatcct g 5117845DNAArtificial SequenceSynthetic oligonucleotide 178gtctggcgat ggtcaataat gtttgggcca cagaagggct tctga 4517945DNAArtificial SequenceSynthetic oligonucleotide 179tcatggcgag ccacctgcat ttacttctgc aagcctgtaa tttac 4518051DNAArtificial SequenceSynthetic oligonucleotide 180aactatcatc ggggccaagg tggcatcatt agtcttcttc cacttttctg g 5118148DNAArtificial SequenceSynthetic oligonucleotide 181tggtgagcac ttaggccctt ccccacctta tgcctcttac cagtaggg 4818248DNAArtificial SequenceSynthetic oligonucleotide 182cacgtaacat cgtgattact gatttgttac ggttttgatt tttttttc 4818344DNAArtificial SequenceSynthetic oligonucleotide 183gatggtcgtc ttagttttaa gatcgtaaaa gatattgctt gtaa 4418445DNAArtificial SequenceSynthetic oligonucleotide 184tggctcgata actttgctga gagtatgtat catgactgca tactg 4518545DNAArtificial SequenceSynthetic oligonucleotide 185cttcttcggt tgcatctgcc cctgaggctc ctctacagag ctcag 4518646DNAArtificial SequenceSynthetic oligonucleotide 186tctctcgttg gaattgaaag aagttttgaa gaaatattaa aagtgc 4618748DNAArtificial SequenceSynthetic oligonucleotide 187gaccgatggt ccttgtgctt tcatgtctct cgggcagata cctaggag 4818845DNAArtificial SequenceSynthetic oligonucleotide 188gatgcgcaca tgaaccacgt cagaagatcc cgggtacctc tctct 4518948DNAArtificial SequenceSynthetic oligonucleotide 189tgactgaagc ccgagtccca agggcagacc cgtgctggga ggaaagtt 4819045DNAArtificial SequenceSynthetic oligonucleotide 190tggcgttcta ggaggtcaaa ataccaatat taagaggagt ttgga 4519147DNAArtificial SequenceSynthetic oligonucleotide 191tccatacctg cattcatcaa ctcctactgt caggaaatat ttactga 4719250DNAArtificial SequenceSynthetic oligonucleotide 192tgttaggctc tgcgtgtctg ttaggagcaa aaggggtgag aaccggccac 5019347DNAArtificial SequenceSynthetic oligonucleotide 193ggttctcgat attttctgtt ttcatgcata tcagcccaac ttgcaat 4719446DNAArtificial SequenceSynthetic oligonucleotide 194tggaactacc gaaaagaaaa atgacaggaa agtgtgggta gtataa

4619541DNAArtificial SequenceSynthetic oligonucleotide 195ctccattgca gagtttgtat atcggtatta tcagacgcag a 4119647DNAArtificial SequenceSynthetic oligonucleotide 196agaatcaagg ccaatagcat tcaaatgtgc ttcatgttat tcatctt 4719747DNAArtificial SequenceSynthetic oligonucleotide 197aataatcgta cattatgcta attacactga tcactataca ttatatg 4719849DNAArtificial SequenceSynthetic oligonucleotide 198acctacctct ccaatccagg aaataaggag ctcccactaa catgtacgg 4919948DNAArtificial SequenceSynthetic oligonucleotide 199tcacaggtcg taagacttaa aactccaggc aactggagag cagtggcc 4820048DNAArtificial SequenceSynthetic oligonucleotide 200tggctcaacc atacagcaca actgggcagt gcctactcaa cagccaag 4820152DNAArtificial SequenceSynthetic oligonucleotide 201agtaaggaat caaggagcag cgcatcctga accatgtgct gcagcatgcg ga 5220245DNAArtificial SequenceSynthetic oligonucleotide 202ataaggtggg tgggacggac tgtgcctttg ttggttggtg gagct 4520349DNAArtificial SequenceSynthetic oligonucleotide 203tacttctcta acgtcaccac cgcatcacgt gggccccttt gggtctgtc 4920445DNAArtificial SequenceSynthetic oligonucleotide 204gtacgccacc aaaatgagtt catgtccagt caactgtatg gaata 4520543DNAArtificial SequenceSynthetic oligonucleotide 205gaagatgccc tctggacctt aaggacattt aaaccccatt ttc 4320647DNAArtificial SequenceSynthetic oligonucleotide 206tgttggattc gatattaata aaagattgta cattcttttt ttttcac 4720746DNAArtificial SequenceSynthetic oligonucleotide 207caggcgagac tgggccctgg aggaaagggg gccccgggtc agggag 4620845DNAArtificial SequenceSynthetic oligonucleotide 208cagcttctca caatttgatt ggatggtaat tgtgaagatc atatt 4520948DNAArtificial SequenceSynthetic oligonucleotide 209ggagatatca cgtgtctctt tgtctggggt gcgtattgag caaggtaa 4821045DNAArtificial SequenceSynthetic oligonucleotide 210tgtaagctca cactgacatg ccgcacatca ctggttagat ccaac 4521153DNAArtificial SequenceSynthetic oligonucleotide 211cttatacctc ggcaaatgtt gcagagatct ggggagcatc acagatgaga agc 5321250DNAArtificial SequenceSynthetic oligonucleotide 212ctactaatag aggcctctgg gccgccaccc tgcgcgcccc tgccgcccgg 5021341DNAArtificial SequenceSynthetic oligonucleotide 213gtccctgctt cccttgaggc cttgctgggc cagcagccta g 4121452DNAArtificial SequenceSynthetic oligonucleotide 214taattggtgg ctatcatgag caatagactt acacatacct caacaccacc cc 5221553DNAArtificial SequenceSynthetic oligonucleotide 215gtactgccag aaacatgtca ctgtgctgtg acatactttt ttgttagttt atg 5321645DNAArtificial SequenceSynthetic oligonucleotide 216tataagggct tttgcaggtg tgggggcagg gtcattgggg ggtgt 4521745DNAArtificial SequenceSynthetic oligonucleotide 217ggatggactt gattagataa ggcctcgaca tagtggctaa tgagc 4521839DNAArtificial SequenceSynthetic oligonucleotide 218cacctcgaag ggtgatgttt ttatgttggg tgatgtctt 3921944DNAArtificial SequenceSynthetic oligonucleotide 219gtcatgcgaa gtctctagac ttttccaaca aggggctttt gaca 4422049DNAArtificial SequenceSynthetic oligonucleotide 220ctctatgcaa ggtactgggg acttctgacc ctctcctcca ttgctagat 4922148DNAArtificial SequenceSynthetic oligonucleotide 221gcttaagatg tgcaatgaga tattttggga aatgggcagt aatgatca 4822244DNAArtificial SequenceSynthetic oligonucleotide 222aggtaacgtt ttactgattc atatgtctac tttgtttcac tgct 4422351DNAArtificial SequenceSynthetic oligonucleotide 223gtgtgtagtt aactcaacct ttttaatgaa acaggagaaa tagacattta c 5122446DNAArtificial SequenceSynthetic oligonucleotide 224gaccgaactc acctttctga aggacctgca ctctcccagg tatacc 4622548DNAArtificial SequenceSynthetic oligonucleotide 225ttatcggact gtaagtgtga ttcaccatat aaacagaatt aaaaacaa 4822648DNAArtificial SequenceSynthetic oligonucleotide 226agatgattct atttgggaag actgactctc tgttacaccc aagggcct 4822754DNAArtificial SequenceSynthetic oligonucleotide 227ttataattcg ttattattaa gattttttta acaggaaaaa agagtattca ctga 5422846DNAArtificial SequenceSynthetic oligonucleotide 228gcagtggtta tcacaataaa ttttttgttt tatatatgaa attagt 4622961DNAArtificial SequenceSynthetic oligonucleotide 229aaataatcag gagaaggaga aggcatgttt gttggtgatt ccaaggagct cgaagcatag 60g 6123064DNAArtificial SequenceSynthetic oligonucleotide 230ggagcagcgt ctctgccatc gtcctcatcc atgtcctggt cacacttcca agagtgtatt 60tagt 6423162DNAArtificial SequenceSynthetic oligonucleotide 231aggtaaatat ttaccacgtc ttggtgttta ttttaccgtc tatatacaag atcatacatt 60ac 6223260DNAArtificial SequenceSynthetic oligonucleotide 232ctttcagtca gatgtatatg catttggaat tgttctgtat gaattgatga aacggagaat 6023361DNAArtificial SequenceSynthetic oligonucleotide 233tatctgctaa gaaacaggca tccatataca gagatgaaaa tgatgatttt gataacctat 60a 6123465DNAArtificial SequenceSynthetic oligonucleotide 234atcttgcctt gccttccacc gtaataccag cataatctac taaagcttct actaatacac 60acatc 6523560DNAArtificial SequenceSynthetic oligonucleotide 235tgtccagtga tatggttatc atgtgaaaca aaactcacct gggtcacttt cgactagaag 6023662DNAArtificial SequenceSynthetic oligonucleotide 236attaaaggcg ccgccgggcg gctcccgctg acatcctggc tctcctgcgc ttcgcgcgaa 60tg 6223760DNAArtificial SequenceSynthetic oligonucleotide 237gatttgcgtt ctgcactatg acatcatttg gccttcaggg tgagttgttt attgcagtat 6023858DNAArtificial SequenceSynthetic oligonucleotide 238agacttcacc ttgtgatctg cagggactga ccttggtgtt gttgtgttgg ccgaaacc 5823958DNAArtificial SequenceSynthetic oligonucleotide 239agagatctcc aaagacactc cacggaatga gggctcgttg cccttgtctt gcgtgatt 5824060DNAArtificial SequenceSynthetic oligonucleotide 240cctatacaat tgagatggtg ggggaaccac aacataactc attcagactg cggacaattc 6024159DNAArtificial SequenceSynthetic oligonucleotide 241tttttattgt atatgcatgc gcatcccaag gaccaagaga ccagctacac cgcttaggt 5924261DNAArtificial SequenceSynthetic oligonucleotide 242gcaggcatca aagtgcacga cgtccggctg aatggctccg cagctggcca atgtacttag 60t 6124359DNAArtificial SequenceSynthetic oligonucleotide 243tctgcattcc ctgtcaccgc gtcactggcc ttcagacaga gccaaggtgc ctgcttgtg 5924462DNAArtificial SequenceSynthetic oligonucleotide 244gcccaagtgt ttctctggca tctgatggtg tctggatcca ccactctact atgcttgaaa 60tc 6224565DNAArtificial SequenceSynthetic oligonucleotide 245gttcagattt cactgcctca tgttgatgtt tctttccaga tgacgactgt cttaatacat 60ggttc 6524661DNAArtificial SequenceSynthetic oligonucleotide 246tagacacatt gtcatcatgg acgggcggtg gatacgtgcc gtctagctga gtcttcataa 60t 6124762DNAArtificial SequenceSynthetic oligonucleotide 247gcttgtcttt gaaacttctt ggcaagtcgg ttaagatctg ggaatcgacg gatattggaa 60aa 6224861DNAArtificial SequenceSynthetic oligonucleotide 248tacctcccat attggggcct acaaaacaaa ttatatcaga aagcaagatt atgatcacaa 60a 6124959DNAArtificial SequenceSynthetic oligonucleotide 249tgccaatgac cacagtgtcg ggcccggcat ccagtgacga gggcgtggtg ggctcgatt 5925060DNAArtificial SequenceSynthetic oligonucleotide 250atccccctta aaatcacact cacttgccgc gcataggcca tctcgatgtt ggctggacta 6025160DNAArtificial SequenceSynthetic oligonucleotide 251ggggaggtga agctgtccat ctcctacaaa aacaataaac tcttcatcat attctcaact 6025263DNAArtificial SequenceSynthetic oligonucleotide 252cgagattttt ttccttccaa tatattctac gtaagttccc ggaaagtccc tatgatcgaa 60atc 6325358DNAArtificial SequenceSynthetic oligonucleotide 253cttttcctta aaaagaaaaa gaaagggagt cattaagcaa ccacttatca cacgtatg 5825460DNAArtificial SequenceSynthetic oligonucleotide 254tgtgtgttat gatttctctt gcagagttgt gaaaaccgtg gctgtgaaaa gagctctgta 6025561DNAArtificial SequenceSynthetic oligonucleotide 255ggccctgcaa atgccctcag cagaagcccc gttgccctcc tggagtgagc aagcaagagt 60c 6125660DNAArtificial SequenceSynthetic oligonucleotide 256gctactccag gcacacgtcg cacatcctgc aggcagagag taagaggaag tcacgcaaac 6025755DNAArtificial SequenceSynthetic oligonucleotide 257gatctctcct gagtcctcac taacaacagg gggttgattt attgttttca agagc 5525854DNAArtificial SequenceSynthetic oligonucleotide 258ccactagccc tggttcaggt cagggatgcc atgttgtcgg ggcccaggca aata 5425953DNAArtificial SequenceSynthetic oligonucleotide 259tggcagcctc actgtgcgga gcatggagcc acacgtggtg taggcacagt ggt 5326058DNAArtificial SequenceSynthetic oligonucleotide 260aagtacccca aagtgtgagg gccttccctc tgccacacat catcgagaac ttatggct 5826155DNAArtificial SequenceSynthetic oligonucleotide 261gtgcttctga aactgttatc ttcccaggag caatttgggg atagaaagca tcagt 5526256DNAArtificial SequenceSynthetic oligonucleotide 262gggccagtct ttaaatgctt cctggaaaat gttgctacct atgaaataaa ccttgt 5626357DNAArtificial SequenceSynthetic oligonucleotide 263aaggaagcag cgtgcagtgc cattccttcc tccaggtaag cctcgttagg gttaggt 5726458DNAArtificial SequenceSynthetic oligonucleotide 264attgggggta tattggaaaa gtatttttgg tgttgaagtt tgggctgtga gccaagct 5826556DNAArtificial SequenceSynthetic oligonucleotide 265gggatgtttc ttgtcctcgt tcaagacaga attcgagagt gagccagtgg ggagtc 5626657DNAArtificial SequenceSynthetic oligonucleotide 266tttgacacca ataaaatgga gtgccactga agggttttga gctgaggaga gaacgaa 5726757DNAArtificial SequenceSynthetic oligonucleotide 267accgtacctc tgcccgacgt gggcaggcgt gagttgtcag tttagagaac attagag 5726855DNAArtificial SequenceSynthetic oligonucleotide 268ctgcttctag ggttgggaac tcccagggaa gaccgggctt gccatgtgca cgaca 5526957DNAArtificial SequenceSynthetic oligonucleotide 269gtcattttgc tgtttgtttt ctatatgcgg tataacattt ttgtctctta ggagaga 5727054DNAArtificial SequenceSynthetic oligonucleotide 270taggagacag agaatgttct gtgggaccac aaccaagaca gaagagctct ccgt 5427159DNAArtificial SequenceSynthetic oligonucleotide 271cctgtaacac acgcccacag gggcttcagg aactgtaaac attcaccctt tatagtcag 5927257DNAArtificial SequenceSynthetic oligonucleotide 272tatttgataa attaacccta gaacaactat ctgcgctcag aaacacatac catcctc 5727357DNAArtificial SequenceSynthetic oligonucleotide 273gagcatcctg aagcaattct gtttgtaatc ctgggagtag taactgggaa tgagatt 5727457DNAArtificial SequenceSynthetic oligonucleotide 274tgaattattt ttcttccctt tcatttttgt ttaagctcta ttgttttttg ttatgga 5727558DNAArtificial SequenceSynthetic oligonucleotide 275agaacaatgt ccacatgttt cctctgtgcc attattaaga tggtcccctt gccatcac 5827655DNAArtificial SequenceSynthetic oligonucleotide 276catcccaccc tgtctcactg gagccaggat ccataaggtc ccgtgagcta gcttg 5527756DNAArtificial SequenceSynthetic oligonucleotide 277tccatcctaa aggacttacg gtttcttaga ataacatgga gtgatttttc agagta 5627856DNAArtificial SequenceSynthetic oligonucleotide 278gaaaacagtc aaaatggctg tcaacaatga aatggataca tcagttgaga gaccat 5627957DNAArtificial SequenceSynthetic oligonucleotide 279gaaagactaa taattttgcc catgatcacc tcactatctc actctgaaat ggcacag 5728054DNAArtificial SequenceSynthetic oligonucleotide 280agtagtcggc atggtgctga gcatcctccg ggaaccgtcc ctacctgaaa ggca 5428158DNAArtificial SequenceSynthetic oligonucleotide 281aattctagct ccaaaatctg ggctcctgac cacagtgtta gaggtgcctt tacgattg 5828256DNAArtificial SequenceSynthetic oligonucleotide 282gaaccgtcac caggtccttt attgcctctt ccaataatag aaattatcca tccgtg 5628359DNAArtificial SequenceSynthetic oligonucleotide 283agtcctaacc taggttacag cccatcacag ctggggcagg tgatgcttag ttatgtaat 5928451DNAArtificial SequenceSynthetic oligonucleotide 284gtcaggctta agaggcaggg ccacctaaac gtctgctgag aacacagggt t 5128557DNAArtificial SequenceSynthetic oligonucleotide 285ttcagatatg actagggaat gtttagaaag tacaggccac atgcttcatg agtagta 5728655DNAArtificial SequenceSynthetic oligonucleotide 286ctccaggtat agatgcaagt aggctggtag atttgatgag gagacaaact tcgga 5528755DNAArtificial SequenceSynthetic oligonucleotide 287ctgggtgtaa agtttctgtg caaacctttg ctacggtgcg tgccgagtca cgcgt 5528859DNAArtificial SequenceSynthetic oligonucleotide 288gacctgtagt cacaagtgta gagagtttga gctttgactt agaaaggcac attagtcta 5928958DNAArtificial SequenceSynthetic oligonucleotide 289ttcactggcg atcaacagta actaataaaa ttcactcatg aatcaacagt acatgcag 5829055DNAArtificial SequenceSynthetic oligonucleotide 290tctctgtagt caatttgatt tttatcaagt tgcattaaat attttaagac ggtag 5529158DNAArtificial SequenceSynthetic oligonucleotide 291cagcctgtgt tcaggatctc acagagtctc tcatgaaaat agttgtgggc ataagcca 5829257DNAArtificial SequenceSynthetic oligonucleotide 292ttgttctcat ctctcagatg cccttctgtg gcccaaacat tattgaccat atcctaa 5729357DNAArtificial SequenceSynthetic oligonucleotide 293gaagcctagg tatgtaaatt ataggcttgc agaagtaaat gcaggtggct ttcgatt 5729459DNAArtificial SequenceSynthetic oligonucleotide 294ggacgagccc cagaaaagtg gaagaagact aatggtgcca ccttggcccc catactctt 5929560DNAArtificial SequenceSynthetic oligonucleotide 295tgccctcggc cctactggta agaggcataa ggtggggaag ggcctaagtg aattattcat 6029658DNAArtificial SequenceSynthetic oligonucleotide 296cttaaaacta aaacaggaaa aaaaaatcaa aaccataaca aatcagtaat tggatcca 5829756DNAArtificial SequenceSynthetic oligonucleotide 297aaatcagtaa aatgtttaca agcaatatct tttatgatct taaaactaag agcgct 5629857DNAArtificial SequenceSynthetic oligonucleotide 298ctacataaca gaattcagta tgcagtcatg atacgtactc tcagcaaagt tgagttc 5729955DNAArtificial SequenceSynthetic oligonucleotide 299tcctctcagt ctctgagctc tgtagaggag cctcgggggc agatgcaacc ttaga 5530055DNAArtificial SequenceSynthetic oligonucleotide 300acacaaaact aaaagcactt ttaatatttc ttcagaactt ctttcaattc ggaga 5530155DNAArtificial SequenceSynthetic oligonucleotide 301agccactcca ctcctaggta tctgcccaag agacatgaaa gcacaaggac actgg 5530255DNAArtificial SequenceSynthetic oligonucleotide 302tgatccccaa cagagagagg tacctgggat cttctgacgt ggttcatgtg tgtca 5530357DNAArtificial SequenceSynthetic oligonucleotide 303tccgaattct ccaactttcc tcccagcaag ggtctgccct tgggactcgg gcgatga 5730458DNAArtificial SequenceSynthetic oligonucleotide 304tttcctttct ttcttccaaa ctcctgttaa tattggtatt ttgacctcct aactgtgg 5830555DNAArtificial SequenceSynthetic oligonucleotide 305acatagaagg tgttcagtaa atatttcctg actgtaggag ttgatgaatg tgcgt 5530655DNAArtificial SequenceSynthetic oligonucleotide 306tcatggccgg tggccggttc tcaccccttt tgcttctaac agacacgcag agcga 5530756DNAArtificial SequenceSynthetic oligonucleotide 307ggagatactg acaattgcaa gttgggctga tatgtatgaa aacagaaaat acggca 5630856DNAArtificial SequenceSynthetic oligonucleotide 308taacaaagac tagcttatac tacccacgct ttcctgtcat ttttcttttc ggttca 5630956DNAArtificial SequenceSynthetic oligonucleotide 309aacattttgt tttataatct gcgtctgata atactgatat acaaactctg ctctag 5631057DNAArtificial SequenceSynthetic oligonucleotide 310agatggtgaa gtaaagatga ataacatgaa gcacgtttga atgctattgg cttctga 5731157DNAArtificial SequenceSynthetic oligonucleotide 311tagtgatatt tcaatacata taatgtatag ttatcagtgt aattagcata aagtaca 5731257DNAArtificial SequenceSynthetic oligonucleotide 312ctttctctag gtgccgtaca tgttagtggg ggctccttat ttcctggatt gaagctc 5731357DNAArtificial SequenceSynthetic oligonucleotide 313aactctcagt

ttgggccgct gctctccagt tgcctggagt tttaagtctt agacgac 5731455DNAArtificial SequenceSynthetic oligonucleotide 314atggccaagc cttggctgtt gagtaggcag tgcccagttg tgctgtatgg ttgga 5531555DNAArtificial SequenceSynthetic oligonucleotide 315tcccgggctc cgcatgctgc agcacgtggt tcaggatgcg ctgctccttg attca 5531651DNAArtificial SequenceSynthetic oligonucleotide 316gaaggacccc agctccacca accaacaaag gcacggtccg tcccacccac c 5131756DNAArtificial SequenceSynthetic oligonucleotide 317gaaatagacc ctcgacagac ccaaaggggc ccacatgatg cggtggtgac gttaga 5631860DNAArtificial SequenceSynthetic oligonucleotide 318cccagatttt gctaatccat acagttgact ggacatgaac tcattttggt gagacacata 6031962DNAArtificial SequenceSynthetic oligonucleotide 319attctgaaag gaatgaaaat ggggtttaaa tgtctttaag gtccagaggg gtaactatac 60tt 6232057DNAArtificial SequenceSynthetic oligonucleotide 320attctgaaga tttatcatga aaaaaaaaga atgtacaatc ttttattaat aaccgtc 5732159DNAArtificial SequenceSynthetic oligonucleotide 321caacctgccc ctccctgacc cggggccccc ttttctccag ggcccagtct tgaatggaa 5932262DNAArtificial SequenceSynthetic oligonucleotide 322gttgacttct tttaaaatat gatcttcaca attatcatcc aatcaaattg ttagacgatg 60ct 6232356DNAArtificial SequenceSynthetic oligonucleotide 323atagctttac cattttacct tgctcaatac acaccccaga caaagagaca cactca 5632456DNAArtificial SequenceSynthetic oligonucleotide 324tttgttagca gggttggatc taaccagtga tgtgtggcat gtcagtgtga atcctt 5632558DNAArtificial SequenceSynthetic oligonucleotide 325cctcgttacc tgcttctcat ctgtgatgct cccctgatct ctgcaacatt cataatga 5832659DNAArtificial SequenceSynthetic oligonucleotide 326gcctggggcc gggcggcagg ggcgcgcagg gtgggggccc agaggcctct ttagacatg 5932751DNAArtificial SequenceSynthetic oligonucleotide 327gagagagggt gctaggctgc tggcccagca aggcgtcaag ggaagcaggg a 5132856DNAArtificial SequenceSynthetic oligonucleotide 328ggggttgggg gggtggtgtt gaggtatgtg taaggctatt gctcatgata tcgccg 5632960DNAArtificial SequenceSynthetic oligonucleotide 329aggcgggaac ataaactaac aaaaaagtat gtcatagcac agtgacatgt tgagcaacct 6033051DNAArtificial SequenceSynthetic oligonucleotide 330tgatgggagc acacccccca atgaccctgc ccccgcacct gcaaaagccc t 5133156DNAArtificial SequenceSynthetic oligonucleotide 331ttaaagcaca ttaaagctca ttagccacta tgtcaaggcc ttatctaatc gattca 5633259DNAArtificial SequenceSynthetic oligonucleotide 332tagtatatca tataaaaata aagacatcac ccaatataaa aacatcaccc tcggactaa 5933358DNAArtificial SequenceSynthetic oligonucleotide 333atgttgaact cttttgtcaa aagccccttg ttgggaaagt ctagagactt aatggtct 5833459DNAArtificial SequenceSynthetic oligonucleotide 334aaaccgtatg tgatctagca atggaggaga gggtgagaag tccccagtac gagcataac 5933560DNAArtificial SequenceSynthetic oligonucleotide 335tcaaatttcc cgtgatcgtt actgcccatt tcccaaaata tctcattgca tggttgggtt 6033656DNAArtificial SequenceSynthetic oligonucleotide 336ggtcatgata agtaagcagt gaaacaaagt agacgtatga atcagtaaaa aatcga 5633761DNAArtificial SequenceSynthetic oligonucleotide 337cttcactcgc agtaaatgtc tatttctcct gttttattaa aaaggttgag atagtctcac 60t 6133860DNAArtificial SequenceSynthetic oligonucleotide 338ctctgcccac ggtatacctg ggagagtgca ggtctttcag aaaggtgagt ctattattac 6033961DNAArtificial SequenceSynthetic oligonucleotide 339tgatcatatg gtttttgttt ttaattctgt ttatgtggtg aatcacactt caccgaataa 60g 6134058DNAArtificial SequenceSynthetic oligonucleotide 340agatagatga cttagaggcc cttgggtgta acagtgagtc agtcttccca ctcttcat 5834166DNAArtificial SequenceSynthetic oligonucleotide 341aatcttcata aaacctcagt gaatactctt ttttactgtt aaaaaaatct agtaatctat 60tatagt 6634260DNAArtificial SequenceSynthetic oligonucleotide 342cttgcttatg aacactaatt tcatatataa aacagaaaat ttattgtgat ggatcatata 6034343DNAArtificial SequenceSynthetic oligonucleotide 343tgcttcgagc tccttggaat caccaacaaa catgccttct cct 4334449DNAArtificial SequenceSynthetic oligonucleotide 344tacactcttg gaagtgtgac caggacatgg atgaggacga tggcagaga 4934541DNAArtificial SequenceSynthetic oligonucleotide 345tatgatcttg tatatagacg gtaaaataaa caccaagacg t 4134644DNAArtificial SequenceSynthetic oligonucleotide 346tccgtttcat caattcatac agaacaattc caaatgcata taca 4434744DNAArtificial SequenceSynthetic oligonucleotide 347ggttatcaaa atcatcattt tcatctctgt atatggatgc ctgt 4434850DNAArtificial SequenceSynthetic oligonucleotide 348tgtgtattag tagaagcttt agtagattat gctggtatta cggtggaagg 5034944DNAArtificial SequenceSynthetic oligonucleotide 349tagtcgaaag tgacccaggt gagttttgtt tcacatgata acca 4435043DNAArtificial SequenceSynthetic oligonucleotide 350cgaagcgcag gagagccagg atgtcagcgg gagccgcccg gcg 4335145DNAArtificial SequenceSynthetic oligonucleotide 351tgcaataaac aactcaccct gaaggccaaa tgatgtcata gtgca 4535243DNAArtificial SequenceSynthetic oligonucleotide 352tcggccaaca caacaacacc aaggtcagtc cctgcagatc aca 4335342DNAArtificial SequenceSynthetic oligonucleotide 353acgcaagaca agggcaacga gccctcattc cgtggagtgt ct 4235442DNAArtificial SequenceSynthetic oligonucleotide 354tgtccgcagt ctgaatgagt tatgttgtgg ttcccccacc at 4235540DNAArtificial SequenceSynthetic oligonucleotide 355aagcggtgta gctggtctct tggtccttgg gatgcgcatg 4035645DNAArtificial SequenceSynthetic oligonucleotide 356tacattggcc agctgcggag ccattcagcc ggacgtcgtg cactt 4535745DNAArtificial SequenceSynthetic oligonucleotide 357agcaggcacc ttggctctgt ctgaaggcca gtgacgcggt gacag 4535848DNAArtificial SequenceSynthetic oligonucleotide 358tcaagcatag tagagtggtg gatccagaca ccatcagatg ccagagaa 4835948DNAArtificial SequenceSynthetic oligonucleotide 359catgtattaa gacagtcgtc atctggaaag aaacatcaac atgaggca 4836044DNAArtificial SequenceSynthetic oligonucleotide 360gaagactcag ctagacggca cgtatccacc gcccgtccat gatg 4436146DNAArtificial SequenceSynthetic oligonucleotide 361caatatccgt cgattcccag atcttaaccg acttgccaag aagttt 4636243DNAArtificial SequenceSynthetic oligonucleotide 362atcataatct tgctttctga tataatttgt tttgtaggcc cca 4336343DNAArtificial SequenceSynthetic oligonucleotide 363gcccaccacg ccctcgtcac tggatgccgg gcccgacact gtg 4336443DNAArtificial SequenceSynthetic oligonucleotide 364cagccaacat cgagatggcc tatgcgcggc aagtgagtgt gat 4336546DNAArtificial SequenceSynthetic oligonucleotide 365agaatatgat gaagagttta ttgtttttgt aggagatgga cagctt 4636646DNAArtificial SequenceSynthetic oligonucleotide 366tcgatcatag ggactttccg ggaacttacg tagaatatat tggaag 4636740DNAArtificial SequenceSynthetic oligonucleotide 367cgtgtgataa gtggttgctt aatgactccc tttctttttc 4036843DNAArtificial SequenceSynthetic oligonucleotide 368gagctctttt cacagccacg gttttcacaa ctctgcaaga gaa 4336949DNAArtificial SequenceSynthetic oligonucleotide 369cttgcttgct cactccagga gggcaacggg gcttctgctg agggcattt 4937046DNAArtificial SequenceSynthetic oligonucleotide 370gcgtgacttc ctcttactct ctgcctgcag gatgtgcgac gtgtgc 4637143DNAArtificial SequenceSynthetic oligonucleotide 371gctcttgaaa acaataaatc aaccccctgt tgttagtgag gac 4337244DNAArtificial SequenceSynthetic oligonucleotide 372tatttgcctg ggccccgaca acatggcatc cctgacctga acca 4437344DNAArtificial SequenceSynthetic oligonucleotide 373accactgtgc ctacaccacg tgtggctcca tgctccgcac agtg 4437446DNAArtificial SequenceSynthetic oligonucleotide 374agccataagt tctcgatgat gtgtggcaga gggaaggccc tcacac 4637543DNAArtificial SequenceSynthetic oligonucleotide 375actgatgctt tctatcccca aattgctcct gggaagataa cag 4337646DNAArtificial SequenceSynthetic oligonucleotide 376acaaggttta tttcataggt agcaacattt tccaggaagc atttaa 4637746DNAArtificial SequenceSynthetic oligonucleotide 377acctaaccct aacgaggctt acctggagga aggaatggca ctgcac 4637845DNAArtificial SequenceSynthetic oligonucleotide 378agcttggctc acagcccaaa cttcaacacc aaaaatactt ttcca 4537944DNAArtificial SequenceSynthetic oligonucleotide 379gactccccac tggctcactc tcgaattctg tcttgaacga ggac 4438043DNAArtificial SequenceSynthetic oligonucleotide 380ttcgttctct cctcagctca aaacccttca gtggcactcc att 4338146DNAArtificial SequenceSynthetic oligonucleotide 381ctctaatgtt ctctaaactg acaactcacg cctgcccacg tcgggc 4638244DNAArtificial SequenceSynthetic oligonucleotide 382tgtcgtgcac atggcaagcc cggtcttccc tgggagttcc caac 4438345DNAArtificial SequenceSynthetic oligonucleotide 383tctctcctaa gagacaaaaa tgttataccg catatagaaa acaaa 4538442DNAArtificial SequenceSynthetic oligonucleotide 384acggagagct cttctgtctt ggttgtggtc ccacagaaca tt 4238547DNAArtificial SequenceSynthetic oligonucleotide 385ctgactataa agggtgaatg tttacagttc ctgaagcccc tgtgggc 4738640DNAArtificial SequenceSynthetic oligonucleotide 386gaggatggta tgtgtttctg agcgcagata gttgttctag 4038746DNAArtificial SequenceSynthetic oligonucleotide 387aatctcattc ccagttacta ctcccaggat tacaaacaga attgct 4638841DNAArtificial SequenceSynthetic oligonucleotide 388tccataacaa aaaacaatag agcttaaaca aaaatgaaag g 4138946DNAArtificial SequenceSynthetic oligonucleotide 389gtgatggcaa ggggaccatc ttaataatgg cacagaggaa acatgt 4639045DNAArtificial SequenceSynthetic oligonucleotide 390caagctagct cacgggacct tatggatcct ggctccagtg agaca 4539144DNAArtificial SequenceSynthetic oligonucleotide 391tactctgaaa aatcactcca tgttattcta agaaaccgta agtc 4439242DNAArtificial SequenceSynthetic oligonucleotide 392atggtctctc aactgatgta tccatttcat tgttgacagc ca 4239340DNAArtificial SequenceSynthetic oligonucleotide 393ctgtgccatt tcagagtgag atagtgaggt gatcatgggc 4039444DNAArtificial SequenceSynthetic oligonucleotide 394tgcctttcag gtagggacgg ttcccggagg atgctcagca ccat 4439545DNAArtificial SequenceSynthetic oligonucleotide 395caatcgtaaa ggcacctcta acactgtggt caggagccca gattt 4539646DNAArtificial SequenceSynthetic oligonucleotide 396cacggatgga taatttctat tattggaaga ggcaataaag gacctg 4639746DNAArtificial SequenceSynthetic oligonucleotide 397attacataac taagcatcac ctgccccagc tgtgatgggc tgtaac 4639839DNAArtificial SequenceSynthetic oligonucleotide 398aaccctgtgt tctcagcaga cgtttaggtg gccctgcct 3939942DNAArtificial SequenceSynthetic oligonucleotide 399tactactcat gaagcatgtg gcctgtactt tctaaacatt cc 4240042DNAArtificial SequenceSynthetic oligonucleotide 400tccgaagttt gtctcctcat caaatctacc agcctacttg ca 4240143DNAArtificial SequenceSynthetic oligonucleotide 401acgcgtgact cggcacgcac cgtagcaaag gtttgcacag aaa 4340247DNAArtificial SequenceSynthetic oligonucleotide 402tagactaatg tgcctttcta agtcaaagct caaactctct acacttg 4740348DNAArtificial SequenceSynthetic oligonucleotide 403ctgcatgtac tgttgattca tgagtgaatt ttattagtta ctgttgat 4840442DNAArtificial SequenceSynthetic oligonucleotide 404ctaccgtctt aaaatattta atgcaacttg ataaaaatca aa 4240547DNAArtificial SequenceSynthetic oligonucleotide 405tggcttatgc ccacaactat tttcatgaga gactctgtga gatcctg 4740644DNAArtificial SequenceSynthetic oligonucleotide 406ttaggatatg gtcaataatg tttgggccac agaagggcat ctga 4440744DNAArtificial SequenceSynthetic oligonucleotide 407aatcgaaagc cacctgcatt tacttctgca agcctataat ttac 4440850DNAArtificial SequenceSynthetic oligonucleotide 408aagagtatgg gggccaaggt ggcaccatta gtcttcttcc acttttctgg 5040951DNAArtificial SequenceSynthetic oligonucleotide 409atgaataatt cacttaggcc cttccccacc ttatgcctct taccagtagg g 5141042DNAArtificial SequenceSynthetic oligonucleotide 410tggatccaat tactgatttg ttatggtttt gatttttttt tc 4241141DNAArtificial SequenceSynthetic oligonucleotide 411agcgctctta gttttaagat cataaaagat attgcttgta a 4141242DNAArtificial SequenceSynthetic oligonucleotide 412gaactcaact ttgctgagag tacgtatcat gactgcatac tg 4241343DNAArtificial SequenceSynthetic oligonucleotide 413tctaaggttg catctgcccc cgaggctcct ctacagagct cag 4341441DNAArtificial SequenceSynthetic oligonucleotide 414tctccgaatt gaaagaagtt ctgaagaaat attaaaagtg c 4141545DNAArtificial SequenceSynthetic oligonucleotide 415ccagtgtcct tgtgctttca tgtctcttgg gcagatacct aggag 4541644DNAArtificial SequenceSynthetic oligonucleotide 416tgacacacat gaaccacgtc agaagatccc aggtacctct ctct 4441745DNAArtificial SequenceSynthetic oligonucleotide 417tcatcgcccg agtcccaagg gcagaccctt gctgggagga aagtt 4541844DNAArtificial SequenceSynthetic oligonucleotide 418ccacagttag gaggtcaaaa taccaatatt aacaggagtt tgga 4441942DNAArtificial SequenceSynthetic oligonucleotide 419acgcacattc atcaactcct acagtcagga aatatttact ga 4242046DNAArtificial SequenceSynthetic oligonucleotide 420tcgctctgcg tgtctgttag aagcaaaagg ggtgagaacc ggccac 4642143DNAArtificial SequenceSynthetic oligonucleotide 421tgccgtattt tctgttttca tacatatcag cccaacttgc aat 4342242DNAArtificial SequenceSynthetic oligonucleotide 422tgaaccgaaa agaaaaatga caggaaagcg tgggtagtat aa 4242339DNAArtificial SequenceSynthetic oligonucleotide 423ctagagcaga gtttgtatat cagtattatc agacgcaga 3942444DNAArtificial SequenceSynthetic oligonucleotide 424tcagaagcca atagcattca aacgtgcttc atgttattca tctt 4442541DNAArtificial SequenceSynthetic oligonucleotide 425tgtactttat gctaattaca ctgataacta tacattatat g 4142644DNAArtificial SequenceSynthetic oligonucleotide 426gagcttcaat ccaggaaata aggagccccc actaacatgt acgg 4442744DNAArtificial SequenceSynthetic oligonucleotide 427gtcgtctaag acttaaaact ccaggcaact ggagagcagc ggcc 4442845DNAArtificial SequenceSynthetic oligonucleotide 428tccaaccata cagcacaact gggcactgcc tactcaacag ccaag 4542947DNAArtificial SequenceSynthetic oligonucleotide 429tgaatcaagg agcagcgcat cctgaaccac gtgctgcagc atgcgga 4743041DNAArtificial SequenceSynthetic oligonucleotide 430ggtgggtggg acggaccgtg cctttgttgg ttggtggagc t 4143143DNAArtificial SequenceSynthetic oligonucleotide 431tctaacgtca ccaccgcatc atgtgggccc ctttgggtct gtc 4343245DNAArtificial SequenceSynthetic oligonucleotide 432gtgtctcacc aaaatgagtt catgtccagt caactgtatg gatta 4543346DNAArtificial SequenceSynthetic oligonucleotide 433gtatagttac ccctctggac cttaaagaca tttaaacccc attttc 4643441DNAArtificial SequenceSynthetic oligonucleotide 434gacggttatt aataaaagat tgtacattct ttttttttca t 4143549DNAArtificial SequenceSynthetic oligonucleotide 435ttccattcaa gactgggccc tggagaaaag ggggccccgg gtcagggag 4943645DNAArtificial SequenceSynthetic oligonucleotide 436catcgtctaa

caatttgatt ggatgataat tgtgaagatc atatt 4543741DNAArtificial SequenceSynthetic oligonucleotide 437gagtgtgtct ctttgtctgg ggtgtgtatt gagcaaggta a 4143844DNAArtificial SequenceSynthetic oligonucleotide 438aaggattcac actgacatgc cacacatcac tggttagatc caac 4443947DNAArtificial SequenceSynthetic oligonucleotide 439tcattatgaa tgttgcagag atcaggggag catcacagat gagaagc 4744048DNAArtificial SequenceSynthetic oligonucleotide 440gtctaaagag gcctctgggc ccccaccctg cgcgcccctg ccgcccgg 4844139DNAArtificial SequenceSynthetic oligonucleotide 441ccctgcttcc cttgacgcct tgctgggcca gcagcctag 3944246DNAArtificial SequenceSynthetic oligonucleotide 442ggcgatatca tgagcaatag ccttacacat acctcaacac cacccc 4644348DNAArtificial SequenceSynthetic oligonucleotide 443ttgctcaaca tgtcactgtg ctatgacata cttttttgtt agtttatg 4844441DNAArtificial SequenceSynthetic oligonucleotide 444agggcttttg caggtgcggg ggcagggtca ttggggggtg t 4144541DNAArtificial SequenceSynthetic oligonucleotide 445tgaatcgatt agataaggcc ttgacatagt ggctaatgag c 4144636DNAArtificial SequenceSynthetic oligonucleotide 446gtccgagggt gatgttttta tattgggtga tgtctt 3644744DNAArtificial SequenceSynthetic oligonucleotide 447agaccattaa gtctctagac tttcccaaca aggggctttt gaca 4444845DNAArtificial SequenceSynthetic oligonucleotide 448tatgctcgta ctggggactt ctcaccctct cctccattgc tagat 4544945DNAArtificial SequenceSynthetic oligonucleotide 449ccaaccatgc aatgagatat tttgggaaat gggcagtaac gatca 4545041DNAArtificial SequenceSynthetic oligonucleotide 450cgatttttta ctgattcata cgtctacttt gtttcactgc t 4145150DNAArtificial SequenceSynthetic oligonucleotide 451agtgagacta tctcaacctt tttaataaaa caggagaaat agacatttac 5045249DNAArtificial SequenceSynthetic oligonucleotide 452taataataga ctcacctttc tgaaagacct gcactctccc aggtatacc 4945345DNAArtificial SequenceSynthetic oligonucleotide 453tattcggtga agtgtgattc accacataaa cagaattaaa aacaa 4545441DNAArtificial SequenceSynthetic oligonucleotide 454gaagagtggg aagactgact cactgttaca cccaagggcc t 4145551DNAArtificial SequenceSynthetic oligonucleotide 455actataatag attactagat ttttttaaca gtaaaaaaga gtattcactg a 5145645DNAArtificial SequenceSynthetic oligonucleotide 456tatgatccat cacaataaat tttctgtttt atatatgaaa ttagt 4545754DNAArtificial SequenceSynthetic oligonucleotide 457tcgtcggcag cgtcagatgt gtataagaga cagccaagca catggatcag tgtt 5445855DNAArtificial SequenceSynthetic oligonucleotide 458tcgtcggcag cgtcagatgt gtataagaga cagagggaag ggcatatctg gatac 5545953DNAArtificial SequenceSynthetic oligonucleotide 459tcgtcggcag cgtcagatgt gtataagaga cagttcacgc ttacccagga gtt 5346058DNAArtificial SequenceSynthetic oligonucleotide 460tcgtcggcag cgtcagatgt gtataagaga cagaaggtaa ctgtccagtc atcaattc 5846155DNAArtificial SequenceSynthetic oligonucleotide 461tcgtcggcag cgtcagatgt gtataagaga caggctgtgt agtttctaag ggtcg 5546252DNAArtificial SequenceSynthetic oligonucleotide 462tcgtcggcag cgtcagatgt gtataagaga cagcccagac gagtacagct ca 5246355DNAArtificial SequenceSynthetic oligonucleotide 463tcgtcggcag cgtcagatgt gtataagaga cagagaatcc tgatctgact ggctt 5546455DNAArtificial SequenceSynthetic oligonucleotide 464tcgtcggcag cgtcagatgt gtataagaga cagcttgccc gagttctact acaga 5546552DNAArtificial SequenceSynthetic oligonucleotide 465tcgtcggcag cgtcagatgt gtataagaga cagctggctc tgtgcagaac tg 5246650DNAArtificial SequenceSynthetic oligonucleotide 466tcgtcggcag cgtcagatgt gtataagaga caggcccaga tcgtgtgctc 5046751DNAArtificial SequenceSynthetic oligonucleotide 467tcgtcggcag cgtcagatgt gtataagaga caggctggac tggcttcaca a 5146855DNAArtificial SequenceSynthetic oligonucleotide 468tcgtcggcag cgtcagatgt gtataagaga cagctcctcg tggatccaaa attgc 5546951DNAArtificial SequenceSynthetic oligonucleotide 469tcgtcggcag cgtcagatgt gtataagaga cagggtttca agccctctgc a 5147051DNAArtificial SequenceSynthetic oligonucleotide 470tcgtcggcag cgtcagatgt gtataagaga cagcagccca agccattgtc t 5147157DNAArtificial SequenceSynthetic oligonucleotide 471tcgtcggcag cgtcagatgt gtataagaga cagagcctaa gcaatataaa tggctgc 5747254DNAArtificial SequenceSynthetic oligonucleotide 472tcgtcggcag cgtcagatgt gtataagaga cagcagagta gagtggtgga tcca 5447351DNAArtificial SequenceSynthetic oligonucleotide 473tcgtcggcag cgtcagatgt gtataagaga cagtgaacac agcccacctc a 5147452DNAArtificial SequenceSynthetic oligonucleotide 474tcgtcggcag cgtcagatgt gtataagaga cagctctgca ctccatgcca ac 5247556DNAArtificial SequenceSynthetic oligonucleotide 475tcgtcggcag cgtcagatgt gtataagaga cagtggaagc ttttgtagaa gatgca 5647660DNAArtificial SequenceSynthetic oligonucleotide 476tcgtcggcag cgtcagatgt gtataagaga cagcagtgta cagtttagga ctaacaatcc 6047753DNAArtificial SequenceSynthetic oligonucleotide 477tcgtcggcag cgtcagatgt gtataagaga caggtcatca gtggtgagga gga 5347855DNAArtificial SequenceSynthetic oligonucleotide 478tcgtcggcag cgtcagatgt gtataagaga cagccaagct acatcagtga tgtgg 5547952DNAArtificial SequenceSynthetic oligonucleotide 479tcgtcggcag cgtcagatgt gtataagaga cagtgtgcag gcacttacca ag 5248052DNAArtificial SequenceSynthetic oligonucleotide 480tcgtcggcag cgtcagatgt gtataagaga caggaagcca ggcctgaaga aa 5248156DNAArtificial SequenceSynthetic oligonucleotide 481tcgtcggcag cgtcagatgt gtataagaga cagactgaag cagatgttga acaaca 5648254DNAArtificial SequenceSynthetic oligonucleotide 482tcgtcggcag cgtcagatgt gtataagaga cagtcattgg cctcgttttt cagt 5448350DNAArtificial SequenceSynthetic oligonucleotide 483tcgtcggcag cgtcagatgt gtataagaga cagcctggtt gcttggcaca 5048453DNAArtificial SequenceSynthetic oligonucleotide 484tcgtcggcag cgtcagatgt gtataagaga cagctccctc aagacctacg tga 5348556DNAArtificial SequenceSynthetic oligonucleotide 485tcgtcggcag cgtcagatgt gtataagaga caggttaaag acggcacttc caacag 5648650DNAArtificial SequenceSynthetic oligonucleotide 486tcgtcggcag cgtcagatgt gtataagaga cagtcctccg tggctctccc 5048752DNAArtificial SequenceSynthetic oligonucleotide 487tcgtcggcag cgtcagatgt gtataagaga cagagcttgg ggacacctct ga 5248851DNAArtificial SequenceSynthetic oligonucleotide 488tcgtcggcag cgtcagatgt gtataagaga caggatggtt ccagctgcgc t 5148961DNAArtificial SequenceSynthetic oligonucleotide 489tcgtcggcag cgtcagatgt gtataagaga cagtccgata taagttaaca atgcaatgtc 60a 6149069DNAArtificial SequenceSynthetic oligonucleotide 490tcgtcggcag cgtcagatgt gtataagaga cagtgatctt atttatatat tttcagtcat 60ttgtcctac 6949158DNAArtificial SequenceSynthetic oligonucleotide 491tcgtcggcag cgtcagatgt gtataagaga caggctccaa catttcatcc aggatttg 5849258DNAArtificial SequenceSynthetic oligonucleotide 492tcgtcggcag cgtcagatgt gtataagaga caggccatta cacctaagca ccatctac 5849359DNAArtificial SequenceSynthetic oligonucleotide 493tcgtcggcag cgtcagatgt gtataagaga cagaaagcta aagcagagaa tgaagttga 5949460DNAArtificial SequenceSynthetic oligonucleotide 494tcgtcggcag cgtcagatgt gtataagaga cagaatatca tgtcctattt ctcctcagct 6049557DNAArtificial SequenceSynthetic oligonucleotide 495tcgtcggcag cgtcagatgt gtataagaga cagggagctg tgacaatgaa aatgcag 5749652DNAArtificial SequenceSynthetic oligonucleotide 496tcgtcggcag cgtcagatgt gtataagaga cagccgtcac cgtggagttt cc 5249762DNAArtificial SequenceSynthetic oligonucleotide 497tcgtcggcag cgtcagatgt gtataagaga cagtggaaga gcttacattt aagtgattac 60tg 6249857DNAArtificial SequenceSynthetic oligonucleotide 498tcgtcggcag cgtcagatgt gtataagaga cagaaagtgg tggtttttaa ccccttc 5749960DNAArtificial SequenceSynthetic oligonucleotide 499tcgtcggcag cgtcagatgt gtataagaga caggcctata gatggcaaat taagagagca 6050064DNAArtificial SequenceSynthetic oligonucleotide 500tcgtcggcag cgtcagatgt gtataagaga cagaaaaagt gaatcaatag ttgtactagt 60gcta 6450154DNAArtificial SequenceSynthetic oligonucleotide 501tcgtcggcag cgtcagatgt gtataagaga cagatgggaa gggtacgatg ttcc 5450264DNAArtificial SequenceSynthetic oligonucleotide 502tcgtcggcag cgtcagatgt gtataagaga caggattagg ataattttcc agctcaaaga 60aaat 6450366DNAArtificial SequenceSynthetic oligonucleotide 503tcgtcggcag cgtcagatgt gtataagaga cagcctctaa aactagagtg cctatagaat 60ttattg 6650461DNAArtificial SequenceSynthetic oligonucleotide 504tcgtcggcag cgtcagatgt gtataagaga cagagtattt agttaacggt tgttttacgc 60t 6150562DNAArtificial SequenceSynthetic oligonucleotide 505tcgtcggcag cgtcagatgt gtataagaga caggaatttt gatgaaaaca ttcctgctat 60ca 6250661DNAArtificial SequenceSynthetic oligonucleotide 506tcgtcggcag cgtcagatgt gtataagaga cagcacagtg ttctacggta tacaagtatc 60t 6150766DNAArtificial SequenceSynthetic oligonucleotide 507tcgtcggcag cgtcagatgt gtataagaga caggctacct tatagtcttc cctagcttaa 60taattt 6650848DNAArtificial SequenceSynthetic oligonucleotide 508tcgtcggcag cgtcagatgt gtataagaga caggcagggt ggctgcgt 4850959DNAArtificial SequenceSynthetic oligonucleotide 509tcgtcggcag cgtcagatgt gtataagaga caggcttgga atgaaatccc tatccctat 5951052DNAArtificial SequenceSynthetic oligonucleotide 510tcgtcggcag cgtcagatgt gtataagaga cagcagtggt cctgacgttc gg 5251162DNAArtificial SequenceSynthetic oligonucleotide 511tcgtcggcag cgtcagatgt gtataagaga cagcctaata cattaaagca gtcacttttc 60ct 6251250DNAArtificial SequenceSynthetic oligonucleotide 512tcgtcggcag cgtcagatgt gtataagaga cagggctcac gtcatgggca 5051363DNAArtificial SequenceSynthetic oligonucleotide 513tcgtcggcag cgtcagatgt gtataagaga cagagaactc aaacaagatt taaggtctag 60aaa 6351454DNAArtificial SequenceSynthetic oligonucleotide 514tcgtcggcag cgtcagatgt gtataagaga cagcctccac tcaaagtttc tggc 5451555DNAArtificial SequenceSynthetic oligonucleotide 515tcgtcggcag cgtcagatgt gtataagaga cagtggcaca gactttattg gctct 5551658DNAArtificial SequenceSynthetic oligonucleotide 516tcgtcggcag cgtcagatgt gtataagaga cagcagagat catttctatt gccacagg 5851758DNAArtificial SequenceSynthetic oligonucleotide 517tcgtcggcag cgtcagatgt gtataagaga caggtaagcc tagtgcccag tatatcat 5851863DNAArtificial SequenceSynthetic oligonucleotide 518tcgtcggcag cgtcagatgt gtataagaga caggctagtg tacgatatgt gtgtattgat 60taa 6351955DNAArtificial SequenceSynthetic oligonucleotide 519tcgtcggcag cgtcagatgt gtataagaga cagaccctcc tgcttatgtg gttac 5552052DNAArtificial SequenceSynthetic oligonucleotide 520tcgtcggcag cgtcagatgt gtataagaga cagccctggg tcacacacaa ca 5252158DNAArtificial SequenceSynthetic oligonucleotide 521tcgtcggcag cgtcagatgt gtataagaga cagaagtgag tgggaacagt catattga 5852255DNAArtificial SequenceSynthetic oligonucleotide 522tcgtcggcag cgtcagatgt gtataagaga caggcagata ggtacagagg cgtct 5552356DNAArtificial SequenceSynthetic oligonucleotide 523tcgtcggcag cgtcagatgt gtataagaga cagttgactt tccagttccc cactta 5652465DNAArtificial SequenceSynthetic oligonucleotide 524tcgtcggcag cgtcagatgt gtataagaga caggttcttg ggaagttttt gattactaat 60tcaat 6552565DNAArtificial SequenceSynthetic oligonucleotide 525tcgtcggcag cgtcagatgt gtataagaga cagcttttta tatattgcac actctaaaaa 60gaggt 6552662DNAArtificial SequenceSynthetic oligonucleotide 526tcgtcggcag cgtcagatgt gtataagaga caggggaaaa acaaaattgt ctcaaaaaat 60gt 6252751DNAArtificial SequenceSynthetic oligonucleotide 527tcgtcggcag cgtcagatgt gtataagaga caggggcgga tgccattgag t 5152866DNAArtificial SequenceSynthetic oligonucleotide 528tcgtcggcag cgtcagatgt gtataagaga caggcacttc taagttatta tgatagagtg 60atgtac 6652958DNAArtificial SequenceSynthetic oligonucleotide 529tcgtcggcag cgtcagatgt gtataagaga caggcagtaa atcaacccgc tataaacg 5853055DNAArtificial SequenceSynthetic oligonucleotide 530tcgtcggcag cgtcagatgt gtataagaga caggttgccc ttgccaatag tgaaa 5553162DNAArtificial SequenceSynthetic oligonucleotide 531tcgtcggcag cgtcagatgt gtataagaga cagcagctag ttctatattt acagacagag 60ac 6253260DNAArtificial SequenceSynthetic oligonucleotide 532tcgtcggcag cgtcagatgt gtataagaga cagaatcctg tatctagtgc caatctagaa 6053360DNAArtificial SequenceSynthetic oligonucleotide 533tcgtcggcag cgtcagatgt gtataagaga cagagtatct ataatagtgc gtggcacata 6053449DNAArtificial SequenceSynthetic oligonucleotide 534tcgtcggcag cgtcagatgt gtataagaga cagggaagac ccggcggga 4953557DNAArtificial SequenceSynthetic oligonucleotide 535tcgtcggcag cgtcagatgt gtataagaga cagtcctctc ctgcttaatg tagtcac 5753669DNAArtificial SequenceSynthetic oligonucleotide 536tcgtcggcag cgtcagatgt gtataagaga cagatccttc cagagaatac acaaattata 60tgtatatat 6953760DNAArtificial SequenceSynthetic oligonucleotide 537tcgtcggcag cgtcagatgt gtataagaga cagggtacat gaccataata aatcagcagg 6053864DNAArtificial SequenceSynthetic oligonucleotide 538tcgtcggcag cgtcagatgt gtataagaga cagctctctc tactgaattt tgattttcca 60tttc 6453964DNAArtificial SequenceSynthetic oligonucleotide 539tcgtcggcag cgtcagatgt gtataagaga cagacactga gtattcccaa tgtaaagaaa 60taat 6454049DNAArtificial SequenceSynthetic oligonucleotide 540tcgtcggcag cgtcagatgt gtataagaga cagcctgggc cttcgccct 4954151DNAArtificial SequenceSynthetic oligonucleotide 541tcgtcggcag cgtcagatgt gtataagaga cagggcccac tgcactcacc t 5154260DNAArtificial SequenceSynthetic oligonucleotide 542tcgtcggcag cgtcagatgt gtataagaga cagcaggtag ggaaagattt cttaagtgag 6054352DNAArtificial SequenceSynthetic oligonucleotide 543tcgtcggcag cgtcagatgt gtataagaga cagcctgcag cccatccaca ac 5254454DNAArtificial SequenceSynthetic oligonucleotide 544tcgtcggcag cgtcagatgt gtataagaga cagactgtga gaggctcaga agga 5454550DNAArtificial SequenceSynthetic oligonucleotide 545tcgtcggcag cgtcagatgt gtataagaga cagggggtca gcaggtggca 5054661DNAArtificial SequenceSynthetic oligonucleotide 546tcgtcggcag cgtcagatgt gtataagaga caggggagat gaaataagta ccaaaatgag 60t 6154754DNAArtificial SequenceSynthetic oligonucleotide 547tcgtcggcag cgtcagatgt gtataagaga caggcattgc cactttggct ttcg 5454866DNAArtificial SequenceSynthetic oligonucleotide 548tcgtcggcag cgtcagatgt gtataagaga cagcctctct aaaacttgat gattttaaca 60tgtaat 6654950DNAArtificial SequenceSynthetic oligonucleotide 549tcgtcggcag cgtcagatgt gtataagaga caggttccca cagccgtggt 5055061DNAArtificial SequenceSynthetic oligonucleotide 550tcgtcggcag

cgtcagatgt gtataagaga cagaaatata tagagccgca caccaaaaat 60a 6155156DNAArtificial SequenceSynthetic oligonucleotide 551tcgtcggcag cgtcagatgt gtataagaga cagaaaaatg gggcagaatg ttgtca 5655252DNAArtificial SequenceSynthetic oligonucleotide 552tcgtcggcag cgtcagatgt gtataagaga caggagcaag ttcggtctgg ct 5255364DNAArtificial SequenceSynthetic oligonucleotide 553tcgtcggcag cgtcagatgt gtataagaga cagcccattg attaaacaaa tattcactga 60gtac 6455450DNAArtificial SequenceSynthetic oligonucleotide 554tcgtcggcag cgtcagatgt gtataagaga cagcttcggg cctctggacc 5055555DNAArtificial SequenceSynthetic oligonucleotide 555tcgtcggcag cgtcagatgt gtataagaga cagatctccc gtctcatcct gaaac 5555656DNAArtificial SequenceSynthetic oligonucleotide 556tcgtcggcag cgtcagatgt gtataagaga caggtggaag gcatactgag tgaact 5655763DNAArtificial SequenceSynthetic oligonucleotide 557tcgtcggcag cgtcagatgt gtataagaga caggtgtctc cattacattg cttgttttaa 60ttt 6355854DNAArtificial SequenceSynthetic oligonucleotide 558tcgtcggcag cgtcagatgt gtataagaga cagtctgaca tttcacagct ggca 5455953DNAArtificial SequenceSynthetic oligonucleotide 559tcgtcggcag cgtcagatgt gtataagaga caggctccca ccagctactg tga 5356056DNAArtificial SequenceSynthetic oligonucleotide 560tcgtcggcag cgtcagatgt gtataagaga caggttcacc cagaagtcat tccgta 5656157DNAArtificial SequenceSynthetic oligonucleotide 561tcgtcggcag cgtcagatgt gtataagaga caggaggcac agtgctttgt atttgat 5756258DNAArtificial SequenceSynthetic oligonucleotide 562tcgtcggcag cgtcagatgt gtataagaga cagttagcca ctaccttttt ggctacta 5856355DNAArtificial SequenceSynthetic oligonucleotide 563tcgtcggcag cgtcagatgt gtataagaga cagcgtttta cagcaagcga cagaa 5556457DNAArtificial SequenceSynthetic oligonucleotide 564tcgtcggcag cgtcagatgt gtataagaga cagatgtgat gtgctctagg aaaatgc 5756561DNAArtificial SequenceSynthetic oligonucleotide 565tcgtcggcag cgtcagatgt gtataagaga caggccttgt gagaacagac taatacagat 60a 6156653DNAArtificial SequenceSynthetic oligonucleotide 566tcgtcggcag cgtcagatgt gtataagaga cagggtaggt gtggtcaggt cga 5356752DNAArtificial SequenceSynthetic oligonucleotide 567tcgtcggcag cgtcagatgt gtataagaga cagacctccc tcttgggatg ca 5256864DNAArtificial SequenceSynthetic oligonucleotide 568tcgtcggcag cgtcagatgt gtataagaga cagggagaat aatagttaat taatccacga 60agca 6456961DNAArtificial SequenceSynthetic oligonucleotide 569tcgtcggcag cgtcagatgt gtataagaga cagagagtct caattattgc tcagttagga 60t 6157056DNAArtificial SequenceSynthetic oligonucleotide 570tcgtcggcag cgtcagatgt gtataagaga cagggtgacc tgcgttactt gcttat 5657155DNAArtificial SequenceSynthetic oligonucleotide 571gtctcgtggg ctcggagatg tgtataagag acaggagaca ggaaagggaa ggagt 5557253DNAArtificial SequenceSynthetic oligonucleotide 572gtctcgtggg ctcggagatg tgtataagag acagtgtctc caggagcagc ttc 5357355DNAArtificial SequenceSynthetic oligonucleotide 573gtctcgtggg ctcggagatg tgtataagag acagatcaac aacagggacc aggta 5557456DNAArtificial SequenceSynthetic oligonucleotide 574gtctcgtggg ctcggagatg tgtataagag acagtgttct aacaggcacc agaagt 5657553DNAArtificial SequenceSynthetic oligonucleotide 575gtctcgtggg ctcggagatg tgtataagag acagaccact ctggctgcaa agt 5357665DNAArtificial SequenceSynthetic oligonucleotide 576gtctcgtggg ctcggagatg tgtataagag acagaagtta ttgttattct tgatggttct 60tttga 6557761DNAArtificial SequenceSynthetic oligonucleotide 577gtctcgtggg ctcggagatg tgtataagag acaggttcca atgaattcaa ttatgctgtc 60a 6157855DNAArtificial SequenceSynthetic oligonucleotide 578gtctcgtggg ctcggagatg tgtataagag acagcaaatg cgtgtcctca gagtt 5557957DNAArtificial SequenceSynthetic oligonucleotide 579gtctcgtggg ctcggagatg tgtataagag acagtcctag tttcgttgat tgcaagg 5758053DNAArtificial SequenceSynthetic oligonucleotide 580gtctcgtggg ctcggagatg tgtataagag acagtccacc atgggaaacc tgg 5358155DNAArtificial SequenceSynthetic oligonucleotide 581gtctcgtggg ctcggagatg tgtataagag acagttcaca ggggcatgtt ttagc 5558254DNAArtificial SequenceSynthetic oligonucleotide 582gtctcgtggg ctcggagatg tgtataagag acagaaaggc aaagagggct ttgg 5458363DNAArtificial SequenceSynthetic oligonucleotide 583gtctcgtggg ctcggagatg tgtataagag acagctgatc tatgattcta aattttgctg 60tca 6358458DNAArtificial SequenceSynthetic oligonucleotide 584gtctcgtggg ctcggagatg tgtataagag acagaacctt ggagataact ctgaagga 5858554DNAArtificial SequenceSynthetic oligonucleotide 585gtctcgtggg ctcggagatg tgtataagag acaggtctct ggaaacagcc cttc 5458657DNAArtificial SequenceSynthetic oligonucleotide 586gtctcgtggg ctcggagatg tgtataagag acagagattg tgtttatgtt cccagca 5758759DNAArtificial SequenceSynthetic oligonucleotide 587gtctcgtggg ctcggagatg tgtataagag acagaacaac aacaacagaa accagttag 5958856DNAArtificial SequenceSynthetic oligonucleotide 588gtctcgtggg ctcggagatg tgtataagag acaggcacct ttcacaatgg ttaagg 5658961DNAArtificial SequenceSynthetic oligonucleotide 589gtctcgtggg ctcggagatg tgtataagag acagtgatag agtcggtaac aatcttgtaa 60g 6159053DNAArtificial SequenceSynthetic oligonucleotide 590gtctcgtggg ctcggagatg tgtataagag acagcccaat ttgggccatg agt 5359152DNAArtificial SequenceSynthetic oligonucleotide 591gtctcgtggg ctcggagatg tgtataagag acagcagttg tgtccctgac gg 5259258DNAArtificial SequenceSynthetic oligonucleotide 592gtctcgtggg ctcggagatg tgtataagag acaggtttcc ttttactccc tagaggtt 5859357DNAArtificial SequenceSynthetic oligonucleotide 593gtctcgtggg ctcggagatg tgtataagag acagtcatgg tttcatttgt ccctaca 5759451DNAArtificial SequenceSynthetic oligonucleotide 594gtctcgtggg ctcggagatg tgtataagag acagaggaag aggccgaggt g 5159556DNAArtificial SequenceSynthetic oligonucleotide 595gtctcgtggg ctcggagatg tgtataagag acagcccaga acataacgac tcaacc 5659655DNAArtificial SequenceSynthetic oligonucleotide 596gtctcgtggg ctcggagatg tgtataagag acagcacagg gggattatgc ttcac 5559753DNAArtificial SequenceSynthetic oligonucleotide 597gtctcgtggg ctcggagatg tgtataagag acagtgaagg aaggcctgga gaa 5359856DNAArtificial SequenceSynthetic oligonucleotide 598gtctcgtggg ctcggagatg tgtataagag acagcgtttc tcactggtct cagatg 5659953DNAArtificial SequenceSynthetic oligonucleotide 599gtctcgtggg ctcggagatg tgtataagag acagtgaccc ttgccctggt aga 5360052DNAArtificial SequenceSynthetic oligonucleotide 600gtctcgtggg ctcggagatg tgtataagag acagctgccc tggagccact ag 5260153DNAArtificial SequenceSynthetic oligonucleotide 601gtctcgtggg ctcggagatg tgtataagag acagaccacg aacagcagaa gca 5360269DNAArtificial SequenceSynthetic oligonucleotide 602gtctcgtggg ctcggagatg tgtataagag acagtgtgta tcatcatctc taatttaaag 60aaaaagtac 6960350DNAArtificial SequenceSynthetic oligonucleotide 603gtctcgtggg ctcggagatg tgtataagag acagtggcca gccaagggga 5060457DNAArtificial SequenceSynthetic oligonucleotide 604gtctcgtggg ctcggagatg tgtataagag acagccgtgt gctccatctt acaatac 5760552DNAArtificial SequenceSynthetic oligonucleotide 605gtctcgtggg ctcggagatg tgtataagag acagggccca gcgtgtgtat ga 5260660DNAArtificial SequenceSynthetic oligonucleotide 606gtctcgtggg ctcggagatg tgtataagag acagtctcca tttgtagctg aattcttgtc 6060760DNAArtificial SequenceSynthetic oligonucleotide 607gtctcgtggg ctcggagatg tgtataagag acagtgtttt tgttttttta ccactggctc 6060859DNAArtificial SequenceSynthetic oligonucleotide 608gtctcgtggg ctcggagatg tgtataagag acaggccaaa cagtgttttg tagaccatt 5960954DNAArtificial SequenceSynthetic oligonucleotide 609gtctcgtggg ctcggagatg tgtataagag acaggatcag ggggcagaag gatg 5461052DNAArtificial SequenceSynthetic oligonucleotide 610gtctcgtggg ctcggagatg tgtataagag acagccctgc tctgacacca gg 5261165DNAArtificial SequenceSynthetic oligonucleotide 611gtctcgtggg ctcggagatg tgtataagag acagtgttta ctaccaaaat aatcaaaagc 60acaaa 6561251DNAArtificial SequenceSynthetic oligonucleotide 612gtctcgtggg ctcggagatg tgtataagag acagtccttg gcagccgttc c 5161354DNAArtificial SequenceSynthetic oligonucleotide 613gtctcgtggg ctcggagatg tgtataagag acagaacaca cagacaggca ggtt 5461465DNAArtificial SequenceSynthetic oligonucleotide 614gtctcgtggg ctcggagatg tgtataagag acagagtgct caatagttac cataatgcta 60tattg 6561557DNAArtificial SequenceSynthetic oligonucleotide 615gtctcgtggg ctcggagatg tgtataagag acagcctcct ctctgtgtcc atagaac 5761666DNAArtificial SequenceSynthetic oligonucleotide 616gtctcgtggg ctcggagatg tgtataagag acagtcaatg gttttaccat ttaaaaattc 60cctatc 6661757DNAArtificial SequenceSynthetic oligonucleotide 617gtctcgtggg ctcggagatg tgtataagag acagctcagt tgctcagaac aatgtcc 5761857DNAArtificial SequenceSynthetic oligonucleotide 618gtctcgtggg ctcggagatg tgtataagag acagggagtt tcatcaccaa gtccaca 5761963DNAArtificial SequenceSynthetic oligonucleotide 619gtctcgtggg ctcggagatg tgtataagag acagcccttg ctatcaatat tcaaagagag 60aaa 6362053DNAArtificial SequenceSynthetic oligonucleotide 620gtctcgtggg ctcggagatg tgtataagag acaggctcgt aggtgtgcac cat 5362168DNAArtificial SequenceSynthetic oligonucleotide 621gtctcgtggg ctcggagatg tgtataagag acagagaaca ttcaatgata taaaaggaat 60aagagaac 6862253DNAArtificial SequenceSynthetic oligonucleotide 622gtctcgtggg ctcggagatg tgtataagag acagtccttg gagctgacat ggc 5362356DNAArtificial SequenceSynthetic oligonucleotide 623gtctcgtggg ctcggagatg tgtataagag acaggggatc tctcatctca ggcttg 5662451DNAArtificial SequenceSynthetic oligonucleotide 624gtctcgtggg ctcggagatg tgtataagag acagtgtgtg ccctcgaacc g 5162554DNAArtificial SequenceSynthetic oligonucleotide 625gtctcgtggg ctcggagatg tgtataagag acagcgaccc catctctgag tcct 5462657DNAArtificial SequenceSynthetic oligonucleotide 626gtctcgtggg ctcggagatg tgtataagag acagctagga gcagtcaggc ttaagag 5762756DNAArtificial SequenceSynthetic oligonucleotide 627gtctcgtggg ctcggagatg tgtataagag acagtgaaga acatgcttgc catagc 5662855DNAArtificial SequenceSynthetic oligonucleotide 628gtctcgtggg ctcggagatg tgtataagag acaggcacta ttcaggcaaa ggctc 5562957DNAArtificial SequenceSynthetic oligonucleotide 629gtctcgtggg ctcggagatg tgtataagag acagcccaga ggattaagag acatggc 5763062DNAArtificial SequenceSynthetic oligonucleotide 630gtctcgtggg ctcggagatg tgtataagag acagaagctc tagaaaaggc aaaactaaac 60ta 6263164DNAArtificial SequenceSynthetic oligonucleotide 631gtctcgtggg ctcggagatg tgtataagag acagtcttcc tattcagcct ataagtgttt 60ctaa 6463266DNAArtificial SequenceSynthetic oligonucleotide 632gtctcgtggg ctcggagatg tgtataagag acagaaaacg acttacacat acctaaaatg 60aaattt 6663359DNAArtificial SequenceSynthetic oligonucleotide 633gtctcgtggg ctcggagatg tgtataagag acagtttgat ttgggagcaa agaatgagt 5963459DNAArtificial SequenceSynthetic oligonucleotide 634gtctcgtggg ctcggagatg tgtataagag acaggcatct ctatgccaaa ctggtcata 5963563DNAArtificial SequenceSynthetic oligonucleotide 635gtctcgtggg ctcggagatg tgtataagag acagccaaac tacttctttt ctaacagaaa 60gca 6363654DNAArtificial SequenceSynthetic oligonucleotide 636gtctcgtggg ctcggagatg tgtataagag acaggcatct cttgtgtcag ccct 5463759DNAArtificial SequenceSynthetic oligonucleotide 637gtctcgtggg ctcggagatg tgtataagag acagccaggg aaaaaatatg ttcgatgcc 5963870DNAArtificial SequenceSynthetic oligonucleotide 638gtctcgtggg ctcggagatg tgtataagag acaggataac atagtaatga atacatttct 60aaaaccgtaa 7063965DNAArtificial SequenceSynthetic oligonucleotide 639gtctcgtggg ctcggagatg tgtataagag acagtccaga agattagttg aaaatttgag 60tacaa 6564064DNAArtificial SequenceSynthetic oligonucleotide 640gtctcgtggg ctcggagatg tgtataagag acagcgtttt gtcatttttg cagataaatg 60tagt 6464153DNAArtificial SequenceSynthetic oligonucleotide 641gtctcgtggg ctcggagatg tgtataagag acagagcaaa accgcaaccc act 5364262DNAArtificial SequenceSynthetic oligonucleotide 642gtctcgtggg ctcggagatg tgtataagag acagactcat atctcccaac acaaaactaa 60aa 6264355DNAArtificial SequenceSynthetic oligonucleotide 643gtctcgtggg ctcggagatg tgtataagag acagtgaagc gtgaacttcc tcagg 5564454DNAArtificial SequenceSynthetic oligonucleotide 644gtctcgtggg ctcggagatg tgtataagag acagcacctg ggaagagttg gtgt 5464559DNAArtificial SequenceSynthetic oligonucleotide 645gtctcgtggg ctcggagatg tgtataagag acagtgaata tgtcttcagt gcttagcct 5964668DNAArtificial SequenceSynthetic oligonucleotide 646gtctcgtggg ctcggagatg tgtataagag acagcctaaa aatcgttact tctcctttat 60tttttttc 6864765DNAArtificial SequenceSynthetic oligonucleotide 647gtctcgtggg ctcggagatg tgtataagag acaggcctat aacaatgtac tagaaccaag 60tattt 6564851DNAArtificial SequenceSynthetic oligonucleotide 648gtctcgtggg ctcggagatg tgtataagag acaggggtgt agggcagggg t 5164958DNAArtificial SequenceSynthetic oligonucleotide 649gtctcgtggg ctcggagatg tgtataagag acagttcaca agcagttgtt gaaagact 5865063DNAArtificial SequenceSynthetic oligonucleotide 650gtctcgtggg ctcggagatg tgtataagag acagaaaaga ctgtcagtga tatcttaggt 60aga 6365161DNAArtificial SequenceSynthetic oligonucleotide 651gtctcgtggg ctcggagatg tgtataagag acagggccaa cattttgttt tataatctgc 60g 6165264DNAArtificial SequenceSynthetic oligonucleotide 652gtctcgtggg ctcggagatg tgtataagag acagctagga tcaaagaaga atagaaaaag 60tggt 6465370DNAArtificial SequenceSynthetic oligonucleotide 653gtctcgtggg ctcggagatg tgtataagag acagacaata attgtactta tttatggagt 60acatagtgat 7065459DNAArtificial SequenceSynthetic oligonucleotide 654gtctcgtggg ctcggagatg tgtataagag acagcctagg aggtgttcct cactaaaat 5965560DNAArtificial SequenceSynthetic oligonucleotide 655gtctcgtggg ctcggagatg tgtataagag acagactatc tacatcagtg cgagagaaag 6065652DNAArtificial SequenceSynthetic oligonucleotide 656gtctcgtggg ctcggagatg tgtataagag acagtgtgca gagtccccca gg 5265750DNAArtificial SequenceSynthetic oligonucleotide 657gtctcgtggg ctcggagatg tgtataagag acagggcctc cagcacgctc 5065854DNAArtificial SequenceSynthetic oligonucleotide 658gtctcgtggg ctcggagatg tgtataagag acaggggtag attccagggg ctct 5465960DNAArtificial SequenceSynthetic oligonucleotide 659gtctcgtggg ctcggagatg tgtataagag acagggaaca ctatctgaaa tagaccctcg 6066056DNAArtificial SequenceSynthetic oligonucleotide 660gtctcgtggg ctcggagatg tgtataagag acagcctgca ggtctttccg attctg

5666166DNAArtificial SequenceSynthetic oligonucleotide 661gtctcgtggg ctcggagatg tgtataagag acaggatttt aaaaccagag ataattctga 60aaggaa 6666254DNAArtificial SequenceSynthetic oligonucleotide 662gtctcgtggg ctcggagatg tgtataagag acaggctggt cctcactgac atcc 5466353DNAArtificial SequenceSynthetic oligonucleotide 663gtctcgtggg ctcggagatg tgtataagag acagtctaac cccgtcatgc tgc 5366467DNAArtificial SequenceSynthetic oligonucleotide 664gtctcgtggg ctcggagatg tgtataagag acaggtgcaa tgttaacttt attaattagt 60tgacttc 6766558DNAArtificial SequenceSynthetic oligonucleotide 665gtctcgtggg ctcggagatg tgtataagag acagttgctg ttccacaaaa catagctt 5866661DNAArtificial SequenceSynthetic oligonucleotide 666gtctcgtggg ctcggagatg tgtataagag acaggctgat taattaggtg tttgttagca 60g 6166753DNAArtificial SequenceSynthetic oligonucleotide 667gtctcgtggg ctcggagatg tgtataagag acagcctcac cctccatccc tca 5366850DNAArtificial SequenceSynthetic oligonucleotide 668gtctcgtggg ctcggagatg tgtataagag acagcccaag gcgggcacct 5066950DNAArtificial SequenceSynthetic oligonucleotide 669gtctcgtggg ctcggagatg tgtataagag acaggggtgc tgcgcccaga 5067067DNAArtificial SequenceSynthetic oligonucleotide 670gtctcgtggg ctcggagatg tgtataagag acagggataa actagtggtt ctttgatctt 60tatcttt 6767165DNAArtificial SequenceSynthetic oligonucleotide 671gtctcgtggg ctcggagatg tgtataagag acagaacata ttgaacttat ttttaaaagg 60gggag 6567253DNAArtificial SequenceSynthetic oligonucleotide 672gtctcgtggg ctcggagatg tgtataagag acagggggga tctgccatac agc 5367358DNAArtificial SequenceSynthetic oligonucleotide 673gtctcgtggg ctcggagatg tgtataagag acagtcaagc cttgactttt aaagcaca 5867467DNAArtificial SequenceSynthetic oligonucleotide 674gtctcgtggg ctcggagatg tgtataagag acagttgttg ttgtcagctt agaatagtat 60atcatat 6767568DNAArtificial SequenceSynthetic oligonucleotide 675gtctcgtggg ctcggagatg tgtataagag acagcccttt tataatcttg ttacttttat 60gttgaact 6867657DNAArtificial SequenceSynthetic oligonucleotide 676gtctcgtggg ctcggagatg tgtataagag acagggggag agaaaaccgt atgtgat 5767762DNAArtificial SequenceSynthetic oligonucleotide 677gtctcgtggg ctcggagatg tgtataagag acagttttag atctctttca gttttggttt 60gc 6267865DNAArtificial SequenceSynthetic oligonucleotide 678gtctcgtggg ctcggagatg tgtataagag acagggagtt ataaaaaaga acagaaggtc 60atgat 6567952DNAArtificial SequenceSynthetic oligonucleotide 679gtctcgtggg ctcggagatg tgtataagag acaggcctcc ttcactcgca gt 5268052DNAArtificial SequenceSynthetic oligonucleotide 680gtctcgtggg ctcggagatg tgtataagag acaggcacaa gcggtcaaca gc 5268157DNAArtificial SequenceSynthetic oligonucleotide 681gtctcgtggg ctcggagatg tgtataagag acaggggttt atcaaaaggt ttgctgc 5768262DNAArtificial SequenceSynthetic oligonucleotide 682gtctcgtggg ctcggagatg tgtataagag acagcgctaa aaaaggaaga actaggaaag 60at 6268370DNAArtificial SequenceSynthetic oligonucleotide 683gtctcgtggg ctcggagatg tgtataagag acagaatatc tatccagagt atcgattaat 60cttcataaaa 7068465DNAArtificial SequenceSynthetic oligonucleotide 684gtctcgtggg ctcggagatg tgtataagag acagttccta gtacgtcatt tataatgaaa 60attgc 6568578DNAArtificial SequenceSynthetic oligonucleotide 685cagacataaa cagaagcttt agtagattat gctggtatta cggtggaagg caaggcaaga 60tacagacaaa aatcaaaa 7868678DNAArtificial SequenceSynthetic oligonucleotide 686cagacataaa cagaagcttt agtagattat gctggtatta gggtggaagg caaggcaaga 60tacagacaaa aatcaaaa 7868721DNAArtificial SequenceSynthetic oligonucleotide 687aaggcagagc aaatgtacag g 21

Claims

1. A method of detecting the presence of rare sequence variants within a DNA region of interest, the method comprising: (a) amplifying one or more region of interest using polymerase chain reaction (PCR) with primers, each primer comprising a 5' sequence-adaptor region and a 3' gene-specific region, thereby generating double-stranded amplicons; (b) denaturing the double-stranded amplicons, thereby generating single-stranded amplicons; (c) hybridizing the single-stranded amplicons to a mixture of negative-selection Sinks; (d) removing the single-stranded amplicons bound to Sinks; (e) amplifying the remaining single-stranded amplicons by PCR using primers comprising sequencing adaptor sequences; and (f) performing high-throughput DNA sequencing.

2. The method of claim 1, wherein the rare variant is of unknown sequence identity.

3. The method of claim 1, wherein the rare variant is of known sequence identity.

4. The method of claim 3, wherein step (c) further comprises hybridizing the single-stranded amplicons to a mixture of positive-selection Probes.

5. The method of claim 4, wherein the Probes comprise toehold probes, fine-tuned probes, or X-probes.

6. The method of either claim 3 or 4, wherein the Probes and Sinks are thermodynamically competitive.

7. The method of any one of claims 3-6, wherein there is one Probe and one Sink for each rare sequence variant.

8. The method of any one of claims 3-6, wherein there is one Probe for each rare sequence variant.

9. The method of any one of claims 3-6, wherein the Probes comprise Probes having paired probe complement and probe protector oligonucleotides of Table 1.

10. The method of any one of claims 1-9, wherein the Sinks comprise Sinks having paired sink complement and sink protector oligonucleotides of Table 2.

11. The method of either claim 3 or 4, wherein step (d) further comprises collecting amplicons bound to Probes.

12. The method of claim 11, wherein step (d) is performed via streptavidin-coated magnetic beads, collecting is performed using a magnet, and the Probes in step (c) are either directly functionalized with a biotin or hybridized to a universal oligonucleotide functionalized with a biotin.

13. The method of claim 11, wherein step (d) is performed via streptavidin-coated agarose beads, collection is performed using centrifugal force, and the Probes in step (c) are either directly functionalized with a biotin or hybridized to a universal oligonucleotide functionalized with a biotin.

14. The method of any one of claims 11-13, wherein removing the single-stranded amplicons bound to Sinks occurs by way of collecting amplicons bound to Probes.

15. The method of any one of claims 1-14, wherein the hybridization in step (c) is performed at a temperature of between about 15.degree. C. and about 75.degree. C.

16. The method of any one of claims 1-15, wherein the hybridization in step (c) is performed in a buffer with a monovalent cation concentration of between about 50 mM and about 5 M.

17. The method of claim 16, wherein the monovalent cation is sodium.

18. The method of any one of claim 1-15, wherein the hybridization in step (c) is performed in a buffer with a divalent cation concentration of between about 3 mM and about 30 mM.

19. The method of claim 18, wherein the divalent cation is magnesium.

20. The method of any one of claims 1-14, wherein the PCR of step (a) is multiplex PCR when amplifying more than one region of interest.

21. The method of any one of claims 1-20, wherein the PCR of step (a) is carried out for 4-20 cycles.

22. The method of any one of claims 1-20, wherein the PCR of step (a) is carried out for no more than 20 cycles.

23. The method of any one of claims 1-22, wherein step (b) is performed via heat denaturation.

24. The method of claim 23, wherein heat denaturation comprises heating the amplicon mixture to at least 80.degree. C. for at least 2 minutes.

25. The method of any one of claims 1-24, wherein step (b) is performed via DNAse activity and wherein one of the primers in step (a) is modified with either a 5' phosphate functionalization to encourage degradation or a 5' functionalization to inhibit degradation.

26. The method of claim 25, wherein the 5' primer functionalization comprises a phosphorothioate, a 2'-O-methyl group, or a non-natural nucleotide.

27. The method of any one of claims 1-26, wherein the Sinks in step (c) comprise toehold probes, fine-tuned probes, or X-probes.

28. The method of any one of claims 1-27, wherein the removing in step (d) is performed via solid-phase separation.

29. The method of any one of claims 1-11 and 20-28, wherein step (d) is performed via streptavidin-coated magnetic beads, removing is performed using a magnet, and the Sinks in step (c) are either directly functionalized with a biotin or hybridized to a universal oligonucleotide functionalized with a biotin.

30. The method of any one of claims 1-11 and 20-28, wherein step (d) is performed via streptavidin-coated agarose beads, removing is performed using centrifugal force, and the Sinks in step (c) are either directly functionalized with a biotin or hybridized to a universal oligonucleotide functionalized with a biotin.

31. The method of any one of claims 1-30, wherein the primers in step (e) are universal primers.

32. The method of any one of claims 1-31, wherein the primers in step (e) further comprise a sample barcode or index sequence.

33. The method of any one of claims 1-32, wherein the sequencing in step (f) is sequencing-by-synthesis.

34. The method of any one of claims 1-32, wherein the sequencing in step (f) is nanopore sequencing.

35. The method of any one of claims 1-32, wherein the sequencing in step (f) is sequencing-by-hybridization.

36. The method of any one of claims 1-35, further comprising (g) analyzing the DNA sequencing data to calculate the ratio of reads observed for variant sequences as compared to wild-type sequences.

37. The method of claim 33, wherein the sequencing in step (f) is paired-end sequencing.

38. The method of claim 36, wherein the analysis in step (g) does not consider any sequencing read in which the forward read and the reverse read do not perfectly agree on the sequence of the amplicon insert.

39. The method of claim 36, wherein the analysis in step (g) does not consider any sequencing reading in which a read quality score is below 30.

40. The method of any one of claims 1-39, further defined as a method of quantifying the presence of rare sequence variants within a DNA region of interest.